Vaccine

ABSTRACT

The present invention is in the field of pneumococcal capsular saccharide conjugate vaccines. Specifically, the present invention relates to sized Streptococcus pneumoniae serotype 6A capsular polysaccharides, in particular Streptococcus pneumoniae serotype 6A capsular polysaccharides having the average size (e.g. Mw) of the Streptococcus pneumoniae serotype 6A capsular polysaccharide is between 100-1000 kDa, suitably conjugated to a carrier protein.

FIELD OF THE INVENTION

The present invention relates to sized Streptococcus pneumoniae serotype6A capsular polysaccharides, in particular Streptococcus pneumoniaeserotype 6A capsular polysaccharides having an average size (e.g. M_(w))between 100-1000, 110-750, 150-500, 180-600, 210-490, 210-450, 180-400,210-400, 210-370, 220-360, 230-350, 240-340, 240-320, 240-310 or 250-310kDa. It additionally relates to sized Streptococcus pneumoniae serotype6A capsular polysaccharides conjugated to a carrier protein, immunogeniccompositions, vaccines and processes for making the sized Streptococcuspneumoniae serotype 6A capsular polysaccharides. It also relates to theuse of the immunogenic compositions and vaccines of the invention intherapy and methods of immunising against Streptococcus pneumoniaeinfection.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive bacteriumresponsible for considerable morbidity and mortality (particularly ininfants and the elderly), causing invasive diseases such as bacteraemiaand meningitis, pneumonia and other non-invasive diseases, such as acuteotitis media. About 800,000 children die annually due to pneumococcaldisease, especially in emerging countries (O-Brien et al. 2009 Lancet374:893-902). The increasing number of antibiotic-resistant strains(Linares et al. 2010 Cin. Microbiol. Infect. 16:402-410) and theseverity of pneumococcal diseases make vaccination the most effectiveintervention.

The major clinical syndromes caused by S. pneumoniae are widelyrecognized and discussed in all standard medical textbooks (Fedson D S,Muscher D M. In: Plotkin S A, Orenstein W A, editors. Vaccines. 4thedition. Philadelphia WB Saunders Co, 2004a: 529-588). For instance,Invasive pneumococcal disease (IPD) is defined as any infection in whichS. pneumoniae is isolated from the blood or another normally sterilesite (Musher D M. Streptococcus pneumoniae. In Mandell G L, Bennett J E,Dolin R (eds). Principles and Practice of Infectious diseases (5th ed).New York, Churchill Livingstone, 2001, p 2128-2147). Chronic obstructivepulmonary disease (COPD) is recognised as encompassing severalconditions (airflow obstruction, chronic bronchitis, bronchiolitis orsmall airways disease and emphysema) that often coexist (Wilson et al.,Eur. Respir. J. 2001; 17: 995-1007). Patients suffer exacerbations oftheir condition that are usually associated with increasedbreathlessness, and often have increased cough that may be productive ofmucus or purulent sputum (Wilson, Eur Respir J 2001 17:995-1007). COPDis defined physiologically by the presence of irreversible or partiallyreversible airway obstruction in patients with chronic bronchitis and/oremphysema (Standards for the diagnosis and care of patients with chronicobstructive pulmonary disease. American Thoracic Society. Am J RespirCrit Care Med. 1995 November; 152(5 Pt 2):577-121). Exacerbations ofCOPD are often caused by bacterial (e.g. pneumococcal) infection (SethiS, Murphy T F. Bacterial infection in chronic obstructive pulmonarydisease in 2000: a state-of-the-art review. Clin Microbiol Rev. 2001April; 14(2):336-63).

Pneumococcus is encapsulated with a chemically linked polysaccharidewhich confers serotype specificity. There are more than 90 knownserotypes of pneumococci, and the capsule is the principle virulencedeterminant for pneumococci, as the capsule not only protects the innersurface of the bacteria from complement, but is itself poorlyimmunogenic. An anti-polysaccharide antibody level has been regarded aspredictive of the protection against invasive pneumcoccal disease (Jodaret al. Vaccine, (21) 2003, p. 3264-3272). After initial licensure of a7-valent conjugate vaccine containing serotypes 4, 6B, 9V, 14, 18C, 19F,23F (PCV7), two pneumococcal conjugate vaccines (PCVs) designed tobroaden coverage have been licensed. The 10-valent pneumococcalHaemophilus influenzae protein D conjugate vaccine (PCV10) containsserotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F conjugated to nontypeable H.influenzae protein D, plus serotype 18C conjugated to tetanus toxoid andserotype 19F conjugated to diphtheria toxoid. The 13-valent pneumococcalconjugate vaccine (PCV13) contains the PCV7 (4, 6B, 9V, 14, 18C, 19F,23F) serotypes plus serotypes 1, 3, 5, 6A, 7F and 19A, conjugated tocross-reactive material CRM197.

It is an object of the present invention to develop improvedStreptococcus pneumoniae polysaccharides and improved Streptococcuspneumoniae polysaccharide conjugate vaccines.

S. pneumoniae serogroup 6 isolates, including isolates of serotypes 6A,6B, 6C, and 6D, are important because they are commonly found ininfections (Song et al. JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p.1758-1764). WO2009/000826A2 discloses a Streptococcus pneumoniaeserotype 6A capsular polysaccharide having a size 1100-1540 (Da×10³)conjugated to protein D and a Streptococcus pneumoniae serotype 6Bcapsular polysaccharide having a size 1069-1391 (Da×10³) conjugated toprotein D (see Table 2 of WO2009/000826A2). Both the Streptococcuspneumoniae serotype 6A capsular polysaccharide and the Streptococcuspneumoniae serotype 6B capsular polysaccharide described inWO2009/000826A2 were native polysaccharides. The chemical structure ofserotypes 6A and 6B only differs by the link between rhamnose andribitol units. As the structure of polysaccharide 6A is very similar topolysaccharide 6B it was originally thought that a native PS6A(polysaccharide 6A) should be used for conjugation as a native PS6B(polysaccharide 6B) is used for conjugation. In the FinIP trial,PHiD-CV10 containing 6B conjugated to protein D was demonstrated to beeffective against serotype 6B (Palmu et al. Lancet 2013; 381: 214-22).However, surprisingly, the present invention provides a sizedStreptococcus pneumoniae 6A polysaccharide with improved properties. Theinventors have found that by using a Streptococcus pneumoniae serotype6A capsular polysaccharide (PS6A) of a particular size, a conjugate maybe obtained having both high immunogenicity and which is filterable.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 Evaluation of PS6A conjugates in 14-valent (14V) AIPO4formulation in the Balb/c mouse with co-administration of Infanrix™ Hexamodel. ELISA anti-PS6A.

FIG. 2(A) Immunogenicity of PS6A-CRM197 in the Balb/c mouse model ELISAresults

FIG. 2(B) Immunogenicity of PS6A-CRM197 in the Balb/c mouse model OPAresults

FIG. 3(A) Evaluation of PS6A-CRM197 ELISA results

FIG. 3(B) Evaluation of PS6A-CRM197 OPA results

DESCRIPTION OF THE INVENTION

The present invention provides a sized Streptococcus pneumoniae serotype6A capsular polysaccharide. In one aspect, the present inventionprovides a Streptococcus pneumoniae serotype 6A capsular polysaccharidewherein the average size (M_(w)) of the S. pneumoniae serotype 6Apolysaccharide is between 100-1000, 110-750, 150-500, 180-600, 210-490,210-450, 180-400, 210-400, 210-370, 220-360, 230-350, 240-340, 240-320,240-310 or 250-310 kDa.

The term “polysaccharide” throughout this specification refers to acomplex carbohydrate composed of a chain of saccharides joined togetherby glycosidic bonds. The polysaccharide may contain at least 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40 or 50 or more saccharides.

For the purposes of the invention, “native polysaccharide” refers to apolysaccharide that has not been subjected to a process (e.g.post-purification), the purpose of which is to reduce the size of thepolysaccharide. A polysaccharide can become slightly reduced in sizeduring normal purification procedures or by degradation duringconjugation. Such a saccharide is still native. Only if thepolysaccharide has been subjected to sizing techniques would thepolysaccharide not be considered native.

For the purposes of the invention, “sized polysaccharide” refers to apolysaccharide that has been subjected to a process (e.g.post-purification), the purpose of which is to reduce the size of thepolysaccharide. Polysaccharides may be sized by mechanical or chemicalsizing techniques. Mechanical sizing techniques that may be used includehigh pressure techniques (such as microfluidization, Emulsiflex™, highpressure homogenization, or Gaulin homogenization) and sonication.Chemical sizing techniques that may be used include acid hydrolysis(e.g. treatment with acetic acid) or treatment with periodate. The term“periodate” includes both periodate and periodic acid; the term alsoincludes both metaperiodate (IO₄ ⁻) and orthoperiodate (IO₆ ⁵⁻) and thevarious salts of periodate (e.g., sodium periodate and potassiumperiodate). The molecular weight ranges described herein refer topurified polysaccharides before conjugation (e.g. before activationwhere activation is carried out).

For the purposes of the invention, “sized by a factor up to ×2” meansthat the saccharide is subject to a process intended to reduce the sizeof the saccharide but to retain a size more than half the size of thenative polysaccharide. Terms such as “sized by a factor up to” ×3, ×4etc. are to be interpreted in the same way, i.e. the saccharide issubject to a process intended to reduce the size of the polysaccharidebut to retain a size more than a third, a quarter etc., respectively,the size of the native polysaccharide.

The term “Molecular weight” or “average molecular weight” or “averagesize” of a polysaccharide as used herein refers to the weight-averagemolecular weight (M_(w)) of the polysaccharide measured prior toconjugation measured by MALLS (Multi-Angle Laser Light Scattering).

The MALLS technique is known in the art and is typically carried out asdescribed in below. For MALLS analysis of pneumococcal polysaccharides,two columns (TSKG6000 and 5000PWxl) may be used in combination and thepolysaccharides are eluted in 0.2M NaCl. Polysaccharides are detectedusing a light scattering detector (for instance Wyatt Dawn DSP (DigitalSignal Processing) equipped with a 10 mW argon laser at 488 nm) and aninterferometric refractometer (for instance Wyatt Otilab DSP (DigitalSignal Processing) equipped with a P100 cell and a red filter at 498nm).

The laser light scattering detector measures the light intensitiesscattered at 16 angles by the macromolecular solution and on the otherhand, an interferometric refractometer placed on-line allows thedetermination of the quantity of sample eluted. From these intensities,the size and shape of the macromolecules in solution can be determined.

The mean molecular weight in weight (M_(w)) is defined as the sum of theweights of all the species multiplied by their respective molecularweight and divided by the sum of weights of all the species.

a) Weight-average molecular weight: -M_(w)-

$M_{w} = {\frac{\sum{W_{i} \cdot M_{i}}}{\sum W_{i}} = \frac{m_{2}}{m_{1}}}$

b) Number-average molecular weight: -M_(n)-

$M_{n} = {\frac{\sum{N_{i} \cdot M_{i}}}{\sum N_{i}} = \frac{m_{1}}{m_{0}}}$

c) Root mean square radius: -Rw- and r²w is the square radius definedby:

${R^{2}w\mspace{14mu} {or}\mspace{14mu} ( r^{2} )w} = \frac{\sum{m_{i} \cdot r_{i}^{2}}}{\sum m_{i}}$

(-m_(i)- is the mass of a scattering centre i and -r_(i)- is thedistance between the scattering centre i and the center of gravity ofthe macromolecule).

d) The polydispersity is defined as the ratio -M_(w)/M_(n)-.

As used herein, the term “treatment” (including variations thereof, e.g.“treat” or “treated”) means any one or more of the following: (i) theprevention of infection or re-infection, as in a traditional vaccine,(ii) the reduction in the severity of, or, in the elimination ofsymptoms, (iii) the delay in recurrence of symptoms, and (iii) thesubstantial or complete elimination of the pathogen or disorder inquestion in a subject. Hence, treatment may be effected prophylactically(prior to infection) or therapeutically (following infection).

For the purposes of this invention, “treatment or prevention ofexacerbations of COPD” or “reduction in severity of COPD exacerbations”refers to a reduction in incidence or rate of COPD exacerbations (forinstance a reduction in rate of 0.1, 0.5, 1, 2, 5, 10, 20% or more) or areduction in severity of COPD exacerbations (e.g. airflow obstruction,chronic bronchitis, bronchiolitis or small airways disease andemphysema), for instance within a patient group immunized with theimmunogenic compositions or vaccines of the invention.

Sized Streptococcus pneumoniae Serotype 6A Capsular Polysaccharides

Streptococcus pneumoniae serotype 6A capsular polysaccharides may besized by mechanical or chemical sizing techniques. In an embodiment, theStreptococcus pneumoniae serotype 6A capsular polysaccharides of theinvention are sized by a chemical sizing technique. Chemical sizingtechniques that may be used include treatment with acetic acid ortreatment with periodate. In one aspect, the Streptococcus pneumoniaeserotype 6A capsular polysaccharides of the invention are sized bytreatment with periodate. The term “periodate” includes both periodateand periodic acid; the term also includes both metaperiodate (IO₄ ⁻) andorthoperiodate (IO6⁵⁻) and the various salts of periodate (e.g., sodiumperiodate and potassium periodate). In another aspect, the Streptococcuspneumoniae serotype 6A capsular polysaccharides of the invention aresized by treatment with acetic acid. In an embodiment, the Streptococcuspneumoniae serotype 6A capsular polysaccharides of the invention aresized by a mechanical sizing technique, for example using a highpressure technique. High pressure techniques include microfluidization,high pressure homogenization, or Gaulin homogenization. In one aspect,the Streptococcus pneumoniae serotype 6A capsular polysaccharide of theinvention is sized by high pressure homogenization. High pressurehomogenization achieves high shear rates by pumping the process streamthrough a flow path with sufficiently small dimensions. The shear rateis increased by using a larger applied homogenization pressure, andexposure time can be increased by recirculating the feed stream throughthe homogenizer. The technique of high pressure homogenization isdescribed in Cho et al. (Int. J. Mol. Sci. 2014, 15). In another aspectof the invention, the Streptococcus pneumoniae serotype 6A capsularpolysaccharide of the invention is sized by Gaulin homogenization.Gaulin homogenization may be carried out using the technique describedin Lander et al. (Biotechnol. Prog. 2000, 16, 80-85). In another aspect,the Streptococcus pneumoniae serotype 6A capsular polysaccharide of theinvention is sized by microfluidization (for example as described in theExamples below).

Sized Streptococcus pneumoniae serotype 6A capsular polysaccharides ofthe present invention have an average size (M_(w)) between 100-1000,110-750, 150-500, 180-600, 210-490, 210-450, 180-400, 210-400, 210-370,220-360, 230-350, 240-340, 240-320, 240-310 or 250-310 kDa. Sizing is bya factor of no more than ×20, ×10, ×8, ×6, ×5, ×4, ×3 or ×2. Forexample, sizing may be from a factor of between ×2 to ×6, ×2 to ×5, ×2to ×4, or ×3 to ×6, ×3 to ×5 or ×3 to ×4. In one aspect, theStreptococcus pneumoniae serotype 6A capsular polysaccharide of theinvention is sized by a factor of no more than ×5. The molecular weightranges described herein refer to molecular weight of the purifiedStreptococcus pneumoniae serotype 6A capsular polysaccharides beforeconjugation (e.g., before activation where activation is carried out).

In an embodiment, the sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide of the invention is antigenic (as determined byELISA), for example having an antigenicity index of between 70 to 200%,preferably between 90% and 150% (e.g. between 120 and 144%). Asexplained in the Examples below, the antigenicity index is measuredrelative to native Streptococcus pneumoniae serotype 6A capsularpolysaccharide which is assigned an antigenicity index of 100% (alsorepresented as 1.0 in the tables below).

Conjugated Streptococcus pneumoniae Serotype 6A Capsular Polysaccharides

Suitably, the sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide of the invention is conjugated to a carrier protein. Thecarrier protein may be selected from the group consisting of TT (tetanustoxoid), DT (diphtheria toxoid), CRM197, fragment C of TT, PhtD(pneumococcal histidine triad protein D), PhtDE fusions (a fusion ofPhtD and PhtE (pneumococcal histidine protein E) particularly thosedescribed in WO 01/98334 and WO 03/54007), detoxified pneumolysin andprotein D. In one aspect of the invention, the sized Streptococcuspneumoniae serotype 6A capsular polysaccharide is conjugated to acarrier protein selected from the group consisting of TT (tetanustoxoid), DT (diphtheria toxoid), CRM197, fragment C of TT, and PhtD(pneumococcal histidine triad protein D). In another aspect of theinvention, the sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide of the invention is conjugated DT or CRM197. In anotheraspect of the invention, the sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide of the invention is conjugated to CRM197.

CRM197 is a non-toxic form of the diphtheria toxin but isimmunologically indistinguishable from the diphtheria toxin (DT).Genetically detoxified analogues of diphtheria toxin include CRM197 andother mutants described in U.S. Pat. No. 4,709,017, U.S. Pat. No.5,843,711, U.S. Pat. No. 5,601,827, and U.S. Pat. No. 5,917,017. CRM197is produced by C. diphtheriae infected by the nontoxigenic phaseβ197tox-created by nitrosoguanidine mutagenesis of the toxigeniccarynephage b (Uchida et al Nature New Biology (1971) 233; 8-11). TheCRM197 protein has the same molecular weight as the diphtheria toxin butdiffers from it by a single base change in the structural gene. Thisleads to a glycine to glutamine change of amino acid at position 52which makes fragment A unable to bind NAD and therefore non-toxic(Pappenheimer 1977, Ann Rev, Biochem. 46; 69-94, Rappuoli Applied andEnvironmental Microbiology September 1983 p 560-564).

Fragment C of TT is the non-toxic carboxy-terminal fragment of thetetanus toxin heavy chain. Tetanus toxin is a single peptide ofapproximately 150 kDa, which consists of 1315 amino-acid residues.Cleavage of tetanus-toxin by papain yields two fragments; one of them,fragment C, is approximately 50 kDa. Fragment C of TT is furtherdescribed in Neubauer et al. Biochim. Biophys. Acta 1981, 27, 141-148.

In an embodiment, the sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide of the invention is conjugated to the carrierprotein via a linker, for instance a bifunctional linker (having tworeactive ends). The linker is optionally heterobifunctional (havingdifferent reactive groups at either end) or homobifunctional (havingidentical reactive groups at either end of a spacer arm), for example areactive amino group and a reactive carboxylic acid group, 2 reactiveamino groups or two reactive carboxylic acid groups. The linker has forexample between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possiblelinker is ADH (adipic acid dihydrazide). Other linkers includeB-propionamido (WO 00/10599), nitrophenyl-ethylamine (Geyer et al (1979)Med. Microbiol. Immunol. 165; 171-288), haloalkyl halides (U.S. Pat. No.4,057,685), glycosidic linkages (U.S. Pat. No. 4,673,574, U.S. Pat. No.4,808,700), hexane diamine and 6-aminocaproic acid (U.S. Pat. No.4,459,286). In another embodiment, the sized Streptococcus pneumoniaeserotype 6A capsular polysaccharide of the invention is directlyconjugated to the carrier protein. In one aspect, the sizedStreptococcus pneumoniae serotype 6A capsular polysaccharide of theinvention is linked to carrier protein via an isourea link. An isourealink is formed by the reaction between a cyanate ester on polysaccharideand an amino group on the carrier. Reference to an “isourea link” hereinrefers to a stable link.

Immunogenic Compositions

In an embodiment, the present invention provides an immunogeniccomposition comprising a sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide (or conjugate) of the invention.

Typically the immunogenic compositions of the invention will comprisecapsular polysaccharide antigens (suitably conjugated), wherein thepolysaccharides are derived from at least ten serotypes of S.pneumoniae. The number of S. pneumoniae capsular polysaccharides canrange from 10 different serotypes (or valences “v”) to 23 differentserotypes (23v, a 23 valent composition). In one embodiment theimmunogenic composition comprises 10 or more, 11 or more, 12 or more, 13or more, 14 or more, 15 or more or 15 or more capsular polysaccharidesfrom different S. pneumoniae serotypes. In one embodiment, there are 10,11, 12, 13, 14 or 15 different S. pneumoniae serotypes. In anotherembodiment, the immunogenic composition of the invention may compriseconjugated S. pneumoniae polysaccharides and unconjugated S. pneumoniaepolysaccharides. In an embodiment, the total number of saccharideserotypes is less than or equal to 23.

In one embodiment the multivalent pneumococcal vaccine of the inventionwill comprise polysaccharides (suitably conjugated) selected from thefollowing serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, although it isappreciated that one or two other serotypes could be substituteddepending on the age of the recipient receiving the vaccine and thegeographical location where the vaccine will be administered. In anembodiment, the vaccine may be an 11-valent vaccine. For example, a11-valent vaccine may comprise polysaccharides from serotypes 1, 4, 5,6A, 6B, 7F, 9V, 14, 18C, 19F and 23F. In an embodiment, the vaccine maybe an 12-valent or 13-valent vaccine. A 12 or 13-valent paediatric(infant) vaccine may also include the 11 valent formulation supplementedwith serotypes 19A, or 22F, or 15, or 19A and 22F, or 19A and 15, or 22Fand 15, whereas a 13-valent elderly vaccine may include the 11 valentformulation supplemented with serotypes 19A and 22F, 8 and 12F, or 8 and15, or 8 and 19A, or 8 and 22F, or 12F and 15, or 12F and 19A, or 12Fand 22F, or 15 and 19A, or 15 and 22F. In an embodiment, the vaccine maybe a 14-valent or 15-valent vaccine. A 14 or 15-valent paediatricvaccine may include the 11 valent formulation described abovesupplemented with serotypes 3, 19A and 22F; serotypes 8, 19A and 22F;serotypes 12F, 19A and 22F; serotypes 15, 19A and 22F; serotypes 3, 8,19A and 22F; serotypes 3, 12F, 19A and 22F; serotypes 3, 15, 19A and22F. In an embodiment, the vaccine may be a 16-valent vaccine. A 16valent vaccine may include the 11 valent formulation described abovesupplemented with serotypes 3, 15B, 19A, 22F and 23F. A 16 valentvaccine may include the 11 valent formulation described abovesupplemented with serotypes 3, 15B, 19A, 22F and 33F. In an embodiment,the vaccine may be a 19-valent vaccine. A 19 valent vaccine may includethe 11 valent formulation described above supplemented with serotypes 8,10A, 11A, 12F, 15B, 19A, 22F and 23F. A 19 valent vaccine may includethe 11 valent formulation described above supplemented with serotypes 8,10A, 11A, 12F, 15B, 19A, 22F and 33F. In an embodiment, the vaccine maybe a 20-valent vaccine. A 20 valent vaccine may include the 11 valentformulation described above supplemented with serotypes 3, 8, 10A, 11A,12F, 15B, 19A, 22F and 23F. A 20 valent vaccine may include the 11valent formulation described above supplemented with serotypes 3, 8,10A, 11A, 12F, 15B, 19A, 22F and 33F. In an embodiment, the vaccine maybe a 21-valent vaccine.

In one embodiment, the immunogenic composition of the inventioncomprises capsular polysaccharides derived from serotypes 1, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19F and 23F (suitably conjugated). In a furtherembodiment of the invention at least 12 saccharide antigens (suitablyconjugated) are included, for example capsular polysaccharides derivedfrom serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. In afurther embodiment of the invention at least 12 saccharide antigens(suitably conjugated) are included, for example capsular polysaccharidesderived from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F.In a further embodiment of the invention, at least 13 polysaccharideantigens (suitably conjugated) are included, for example a vaccine maycomprise capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6A,6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, although further saccharideantigens, for example 23 valents (such as serotypes 1, 2, 3, 4, 5, 6B,7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15, 17F, 18C, 19A, 19F, 20, 22F, 23Fand 33F), are also contemplated by the invention. In a furtherembodiment of the invention, at least 15 saccharide antigens (suitablyconjugated) are included, for example capsular polysaccharides derivedfrom serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23Fand 33F. In a further embodiment of the invention, at least 15saccharide antigens (suitably conjugated) are included, for examplecapsular polysaccharides derived from serotypes 1, 4, 5, 6A, 6B, 7F, 9V,14, 15B, 18C, 19A, 19F 22F, 23F and 33F. In a further embodiment of theinvention, at least 16 saccharide antigens (suitably conjugated) areincluded, for example capsular polysaccharides derived from serotypes 1,3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F 22F, 23F and 33F. Inanother embodiment, the immunogenic composition comprises (conjugated)capsular (poly)saccharide of serotype 33F of S. pneumoniae. In anotherembodiment, the immunogenic composition comprises (conjugated) capsular(poly)saccharide of serotype 15C of S. pneumoniae. In anotherembodiment, the immunogenic composition comprises (conjugated) capsular(poly)saccharide of serotype 12F of S. pneumoniae. In an embodiment,there are 10 to 23 different S. pneumoniae capsular polysaccharideserotypes (suitably conjugated).

Carrier Proteins

Examples of carrier proteins which may be used in the present inventionare DT (Diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, DT,CRM197 other DT point mutants, such as CRM176, CRM228, CRM 45 (Uchida etal J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103and CRM107 and other mutations described by Nicholls and Youle inGenetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat.No. 4,950,740; mutation of at least one or more residues Lys 516, Lys526, Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat.No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment of DT disclosed inU.S. Pat. No. 5,843,711, pneumococcal pneumolysin (Kuo et al (1995)Infect Immun 63; 2706-13) including pneumolysin (Ply) detoxified in somefashion for example GMBS detoxified pneumolysin (dPly-GMBS) (WO04081515, PCT/EP2005/010258) or formaldehyde detoxified pneumolysin(dPly-formol), Pht (pneumococcal histidine triad) family proteins(PhtX), including PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins forexample PhtDE fusions, PhtBE fusions (WO 01/98334 and WO 03/54007), (PhtA-E are described in more detail below) OMPC (meningococcal outermembrane protein—usually extracted from N. meningitidis serogroupB—EP0372501), Neisseria meningitidis porin PorB, PD (Haemophilusinfluenzae protein D—see, e.g., EP 0 594 610 B), or immunologicallyfunctional equivalents thereof, synthetic peptides (EP0378881,EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussisproteins (WO 98/58668, EP0471177), cytokines, lymphokines, growthfactors or hormones (WO 91/01146), artificial proteins comprisingmultiple human CD4+ T cell epitopes from various pathogen derivedantigens (Falugi et al (2001) Eur J Immunol 31; 3816-3824) such as N19protein (Baraldoi et al (2004) Infect Immun 72; 4884-7) pneumococcalsurface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337),toxin A or B of Clostridium difficile (WO 00/61761). In an embodiment,in the immunogenic composition of the invention each Streptococcuspneumoniae capsular saccharide is conjugated to a carrier proteinindependently selected from the group consisting of DT, CRM 197, TT,Fragment C of TT, dPly (detoxified pneumolysin), PhtA, PhtB, PhtD, PhtE,PhtDE OmpC, PorB and Haemophilus influenzae Protein D. In a furtherembodiment, each Streptococcus pneumoniae capsular saccharide isconjugated to a carrier protein independently selected from the groupconsisting of TT, DT, CRM197, fragment C of TT, PhtD, PhtDE fusions(particularly those described in WO 01/98334 and WO 03/54007),detoxified pneumolysin and protein D. In a further embodiment, eachStreptococcus pneumoniae capsular saccharide is conjugated to a carrierprotein independently selected from the group consisting of TT, DT,CRM197, PhtD, detoxified pneumolysin and protein D. In a furtherembodiment, each Streptococcus pneumoniae capsular saccharide isconjugated to a carrier protein independently selected from the groupconsisting of TT, DT, CRM197, PhtD and protein D. In a furtherembodiment, each Streptococcus pneumoniae capsular saccharide isconjugated to a carrier protein independently selected from the groupconsisting of TT, DT, CRM197 and protein D.

In an embodiment, the immunogenic composition of the invention comprisestwo or more different carrier proteins. In an embodiment, theimmunogenic composition of the invention comprises 2, 3, 4, 5 or 6different carrier proteins. Each type of carrier protein may act ascarrier for more than one polysaccharide, which polysaccharides may befrom the same or different serotypes. In one embodiment, two or moredifferent polysaccharide serotypes may be conjugated to the same carrierprotein, either to the same molecule of carrier protein (carriermolecules having 2 or more different polysaccharide serotypes conjugatedto it) [see for instance WO 04/083251] or to different molecules of thesame carrier protein (each molecule of protein carrier only having oneserotype of saccharide conjugated to it).

In an embodiment, the immunogenic composition of the invention comprisesprotein D from Haemophilus influenzae (PD), for example, protein Dsequence from FIG. 9 (FIGS. 9a and 9b together, 364 amino acids) of EP0594610 (SEQ ID NO: 1). Inclusion of this protein in the immunogeniccomposition may provide a level of protection against Haemophilusinfluenzae related otitis media (Pyrmula et al Lancet 367; 740-748(2006)). Protein D may be used as a full length protein or as a fragment(for example, Protein D may be as described in WO0056360). For example,a protein D sequence may comprise (or consist) of the protein D fragmentdescribed in EP0594610 which begins at the sequence SSHSSNMANT(SerSerHisSerSerAsnMetAlaAsnThr) (SEQ ID NO. 3), and lacks the 19N-terminal amino acids from FIG. 9 of EP0594610, optionally with thetripeptide MDP from NS1 fused to the N-terminal of said protein Dfragment (348 amino acids) (SEQ ID NO:2). In one aspect, the protein Dor fragment of protein D is unlipidated. The protein D could be presentin the immunogenic composition as a free protein or as a carrierprotein. In one aspect, protein D is present in the immunogeniccomposition as free protein. In another aspect, protein D is presentboth as a carrier protein and as free protein. In a further aspect,protein D is present as a carrier protein for one or more of thepolysaccharides. In a further aspect, 2-9 of the capsularpolysaccharides selected from different serotypes are conjugated toprotein D. In a further aspect, protein D is present as a carrierprotein for the majority of the polysaccharides, for example 6, 7, 8, 9or more of the polysaccharides may be conjugated to protein D.

In an embodiment, the immunogenic composition of the invention contains2-8, 2-7, 2-6, 2-5, 3-5, 4-5, 2-4, 2-3, 3-4 or 2, 3, 4, 5, 6, 7 or 8capsular saccharide serotype conjugates in which protein D is thecarrier protein. For example, 2-8, 2-7, 2-6, 2-5, 3-5, 4-5, 2-4, 2-3,3-4 or 2, 3, 4, 5, 6, 7 or 8 polysaccharides selected from serotype 1,3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F are conjugated toprotein D. For example, polysaccharides from serotypes 1, 4, 5, 6B, 7F,9V, 14 and 23F are conjugated to protein D.

In an embodiment, polysaccharides from at least serotypes 1 and 3, 1 and4, 1 and 5, 1 and 6A, 1 and 6B, 1 and 7, 1 and 9V, 1 and 14, 1 and 22F,1 and 23F, 3 and 4, 3 and 5, 3 and 6A, 3 and 6B, 3 and 7F, 3 and 9V, 3and 14, 3 and 22F, 3 and 23F, 4 and 5, 4 and 6A, 4 and 6B, 4 and 7F, 4and 9V, 4 and 14, 4 and 22F, 4 and 23F, 5 and 6A, 5 and 6B, 5 and 7F, 5and 9V, 5 and 14, 5 and 22F, 5 and 23F, 6A and 6B, 6A and 7F, 6A and 9V,6A and 14, 6A and 22F, 6A and 23F, 6B and 7F, 6B and 9V, 6B and 14, 6Band 22F, 6B and 23F, 7F and 9V, 7F and 14, 7F and 22F, 7F and 23F, 9Vand 14, 9V and 22F, 9V and 23F, 14 and 22F, 14 and 23F or 22F and 23Fare conjugated to protein D.

In an embodiment, polysaccharides from at least serotypes 1, 3 and 4; 1,3 and 5; 1, 3 and 6A; 1, 3 and 6B; 1, 3 and 7F; 1, 3 and 9V; 1, 3 and14; 3, 4 and 7F; 3, 4 and 5; 3, 4 and 7F; 3, 4 and 9V; 3, 4 and 14; 4, 5and 7F; 4, 5 and 9V; 4, 5, and 14; 5, 7F and 9V; 5, 7F and 14; 7F, 9Vand 14; 1, 3, 4 and 5; 3, 4, 5 and 7F; 4, 5, 7F and 9V; 4, 5, 7F and 14;4, 5, 9V and 14; 4, 7F, 9V and 14; 5, 7F, 9V and 14; or 4, 5, 7F, 9V and14 are conjugated to protein D.

For example, in a 10 valent S. pneumoniae immunogenic composition, 2, 3,4, 5, 6, 7 or 8 of the capsular polysaccharides from different serotypesare conjugated to protein D. For example, in a 11 valent S. pneumoniaeimmunogenic composition, 2, 3, 4, 5, 6, 7 or 8 of the capsularpolysaccharides from different serotypes are conjugated to protein D.For example, in a 12 valent S. pneumoniae immunogenic composition, 2, 3,4, 5, 6, 7 or 8 of the capsular polysaccharides from different serotypesare conjugated to protein D. For example, in a 13 valent S. pneumoniaeimmunogenic composition, 2, 3, 4, 5, 6, 7 or 8 of the capsularpolysaccharides from different serotypes are conjugated to protein D.For example, in a 14 valent S. pneumoniae immunogenic composition, 2, 3,4, 5, 6, 7 or 8 of the capsular polysaccharides from different serotypesare conjugated to protein D. For example, in a 15 valent S. pneumoniaeimmunogenic composition, 2, 3, 4, 5, 6, 7 or 8 of the capsularpolysaccharides from different serotypes are conjugated to protein D.For example, in a 16 valent S. pneumoniae immunogenic composition, 2, 3,4, 5, 6, 7 or 8 of the capsular polysaccharides from different serotypesare conjugated to protein D. For example, in a 17 valent S. pneumoniaeimmunogenic composition, 2, 3, 4, 5, 6, 7 or 8 of the capsularpolysaccharides from different serotypes are conjugated to protein D.For example, in a 18 valent S. pneumoniae immunogenic composition, 2, 3,4, 5, 6, 7, 8 or 9 of the capsular polysaccharides from differentserotypes are conjugated to protein D. For example, in a 19 valent S.pneumoniae immunogenic composition, 2, 3, 4, 5, 6, 7, 8 or 9 of thecapsular polysaccharides from different serotypes are conjugated toprotein D. Optionally, the serotypes conjugated to protein D areselected from the groups described above.

In an embodiment, the immunogenic composition of the invention comprisesat least one capsular saccharide conjugated to tetanus toxoid (TT). Inanother embodiment, capsular saccharide 18C is conjugated to TT,optionally wherein 18C is the only saccharide in the compositionconjugated to tetanus toxoid (TT).

In an aspect of the present invention, serotype 19F is conjugated to DTor CRM197. In another aspect, serotype 19F is conjugated to DT. In oneaspect, the remaining saccharide serotypes of the immunogeniccomposition may all be conjugated to one or more carrier proteins thatare not DT (i.e. only 19F is conjugated to DT). In one embodiment, 19Fis conjugated to DT or CRM197, and the remaining serotypes areconjugated to carrier proteins independently selected from PhtD, PD(Protein D), TT (Tetanus Toxoid), DT (Diphtheria Toxoid) and CRM197. Inanother embodiment, 19F is conjugated to DT or CRM 197, and theremaining serotypes are conjugated to carrier proteins independentlyselected from PD, TT, DT and CRM197. In a further embodiment, 19F isconjugated to DT or CRM 197, and the remaining serotypes are conjugatedto carrier proteins independently selected from PD, TT and CRM197 (forexample as described in WO2007/071710A2 and WO2007/071707A2).

In an embodiment, the sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide of the invention and Streptococcus pneumoniaeserotype 6B capsular polysaccharide are conjugated to different carrierproteins. In a further embodiment, the sized Streptococcus pneumoniaeserotype 6A capsular polysaccharide of the invention is conjugated toCRM 197. In a further embodiment a Streptococcus pneumoniae serotype 6Bcapsular polysaccharide conjugate is present, but is not conjugated toDT or CRM 197. In a further embodiment, the sized Streptococcuspneumoniae serotype 6A capsular polysaccharide is conjugated to CRM197and Streptococcus pneumoniae serotype 6B capsular polysaccharideStreptococcus pneumoniae serotype is conjugated to a carrier proteinother than CRM197. In a further embodiment, the sized Streptococcuspneumoniae serotype 6A capsular polysaccharide is conjugated to CRM197and Streptococcus pneumoniae serotype 6B capsular polysaccharideStreptococcus pneumoniae serotype is conjugated to a carrier proteinselected from PhtD, PD (Protein D), TT (Tetanus Toxoid) or DT(Diphtheria Toxoid). In a further embodiment, the sized Streptococcuspneumoniae serotype 6A capsular polysaccharide is conjugated to CRM197and Streptococcus pneumoniae serotype 6B capsular polysaccharide isconjugated to protein D.

In an embodiment of the invention, the carrier protein conjugated to oneor more of the S. pneumoniae capsular polysaccharides is a member of thepolyhistidine triad family (Pht) proteins, fragments or fusion proteinsthereof. The PhtA, PhtB, PhtD or PhtE proteins may have an amino acidsequence sharing 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with asequence disclosed in WO 00/37105 or WO 00/39299 (e.g. with amino acidsequence 1-838 or 21-838 of SEQ ID NO: 4 of WO 00/37105 for PhtD). Forexample, fusion proteins are composed of full length or fragments of 2,3 or 4 of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins arePhtA/B, PhtA/D, PhtA/E, PhtB/A, PhtB/D, PhtB/E. PhtD/A. PhtD/B, PhtD/E,PhtE/A, PhtE/B and PhtE/D, wherein the proteins are linked with thefirst mentioned at the N-terminus (see for example WO01/98334).

Where fragments of Pht proteins are used (separately or as part of afusion protein), each fragment optionally contains one or more histidinetriad motif(s) and/or coiled coil regions of such polypeptides. Ahistidine triad motif is the portion of polypeptide that has thesequence HxxHxH where H is histidine and x is an amino acid other thanhistidine. A coiled coil region is a region predicted by “Coils”algorithm Lupus, A et al (1991) Science 252; 1162-1164. In anembodiment, the fragment includes one or more histidine triad motif aswell as at least one coiled coil region. In an embodiment, the fragmentcontains exactly or at least 2, 3, 4 or 5 histidine triad motifs(optionally, with native Pht sequence between the 2 or more triads, orintra-triad sequence that is more than 50, 60, 70, 80, 90 or 100%identical to a native pneumococcal intra-triad Pht sequence—e.g. theintra-triad sequence shown in SEQ ID NO: 4 of WO 00/37105 for PhtD). Inan embodiment, the fragment contains exactly or at least 2, 3 or 4coiled coil regions. In an embodiment, a Pht protein disclosed hereinincludes the full length protein with the signal sequence attached, themature full length protein with the signal peptide (for example 20 aminoacids at N-terminus) removed, naturally occurring variants of Phtprotein and immunogenic fragments of Pht protein (e.g. fragments asdescribed above or polypeptides comprising at least 15 or 20 contiguousamino acids from an amino acid sequence in WO00/37105 (SEQ ID NOs 4, 6,8 or 10) or WO00/39299 (SEQ ID NOs 2, 4, 6, 8, 10 or 14) wherein saidpolypeptide is capable of eliciting an immune response specific for saidamino acid sequence in WO00/37105 or WO00/39299).

In particular, the term “PhtD” as used herein includes the full lengthprotein with the signal sequence attached, the mature full lengthprotein with the signal peptide (for example 20 amino acids atN-terminus) removed, naturally occurring variants of PhtD andimmunogenic fragments of PhtD (e.g. fragments as described above orpolypeptides comprising at least 15 or 20 contiguous amino acids from aPhtD amino acid sequence in WO00/37105 or WO00/39299) wherein saidpolypeptide is capable of eliciting an immune response specific for saidPhtD amino acid sequence in WO00/37105 or WO00/39299 (e.g. SEQ ID NO: 4of WO 00/37105 or SEQ ID NO: 14 of WO 00/39299 for PhtD). All forms ofPhtD mentioned above can be used in the present invention.

Conjugation Processes

The saccharide conjugates present in the immunogenic compositions of theinvention may be prepared by the conjugation methods of the presentinvention or any known coupling technique. The conjugation method mayrely on activation of the saccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Theactivated saccharide may thus be coupled directly or via a spacer(linker) group to an amino group on the carrier protein. For example,the spacer could be cystamine or cysteamine to give a thiolatedpolysaccharide which could be coupled to the carrier via a thioetherlinkage obtained after reaction with a maleimide-activated carrierprotein (for example using GMBS (4-Maleimidobutyric acidN-hydroxysuccinimide ester)) or a haloacetylated carrier protein (forexample using SIAB (succinimidyl (4-iodoacetyl)aminobenzoate), or SIA(succinimidyl iodoacetate), or SBAP(succinimidyl-3-(bromoacetamide)propionate)). In an embodiment, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or ADH (adipic acid dihydrazide) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide (e.g.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC or EDC)) chemistryvia a carboxyl group on the protein carrier. Such conjugates aredescribed in PCT published application WO 93/15760 Uniformed ServicesUniversity and WO 95/08348 and WO 96/29094.

In an embodiment, at least one of the S. pneumoniae capsularpolysaccharides is directly conjugated to a carrier protein (e.g. usingone of the chemistries described above). In an embodiment, at least oneof the S. pneumoniae capsular polysaccharides is directly conjugated by1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). In anembodiment, the majority of the capsular polysaccharides for example 5,6, 7, 8, 9 or more are directly linked to the carrier protein by CDAP(see WO 95/08348 and WO 96/29094)

In an embodiment, the Streptococcus pneumoniae polysaccharide isconjugated to the carrier protein via a linker, for instance abifunctional linker. The linker is optionally heterobifunctional orhomobifunctional, having for example a reactive amino group and areactive carboxylic acid group, 2 reactive amino groups or two reactivecarboxylic acid groups. The linker has for example between 4 and 20, 4and 12, 5 and 10 carbon atoms. A possible linker is ADH (adipic aciddihydrazide). Other linkers include B-propionamido (WO 00/10599),nitrophenyl-ethylamine (Geyer et al (1979) Med. Microbiol. Immunol. 165;171-288), haloalkyl halides (U.S. Pat. No. 4,057,685), glycosidiclinkages (U.S. Pat. No. 4,673,574, U.S. Pat. No. 4,808,700), hexanediamine and 6-aminocaproic acid (U.S. Pat. No. 4,459,286). In anembodiment, the immunogenic composition of the invention may comprise18C capsular polysaccharide conjugated to the carrier protein via alinker, optionally the linker is ADH. In an embodiment, the immunogeniccomposition of the invention may comprise 22F capsular polysaccharideconjugated to the carrier protein via a linker, optionally the linker isADH (for example as described in WO2007/071711A2).

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide,N-hydroxysulfosuccinimide (S-NHS), EDC,0-(N-Succinimidyl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate (TSTU).Many are described in WO 98/42721. Conjugation may involve a carbonyllinker which may be formed by reaction of a free hydroxyl group of thepolysaccharide with Carbonyldiimidazole (CDI) (Bethell et al J. Biol.Chem. 1979, 254; 2572-4, Hearn et al J. Chromatogr. 1981. 218; 509-18)followed by reaction of with a protein to form a carbamate linkage. Thismay involve reduction of the anomeric terminus to a primary hydroxylgroup, optional protection/deprotection of the primary hydroxyl groupreaction of the primary hydroxyl group with CDI to form a CDI carbamateintermediate and coupling the CDI carbamate intermediate with an aminogroup on a protein.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide (or CDAP)activated polysaccharide derivatised with adipic acid dihydrazide (ADH)to the protein carrier by Carbodiimide condensation (Chu C. et alInfect. Immunity, 1983 245 256), for example using EDAC.

In an embodiment, at least one S. pneumoniae polysaccharide isconjugated to a carrier protein via a linker using CDAP and EDAC. Forexample, 18C or 22F may be conjugated to a protein via a linker (forexample those with two hydrazino groups at its ends such as ADH) usingCDAP and EDAC as described above. When a linker is used, CDAP may beused to conjugate the polysaccharide to a linker and EDAC may then beused to conjugate the linker to a protein or, alternatively EDAC may beused first to conjugate the linker to the protein, after which CDAP maybe used to conjugate the linker to the saccharide.

In an embodiment, a hydroxyl group (suitably an activated hydroxyl groupfor example a hydroxyl group activated to make a cyanate ester [e.g.with CDAP]) on a polysaccharide is linked to an amino or carboxylicgroup on a protein either directly or indirectly (through a linker).Where a linker is present, a hydroxyl group on a polysaccharide issuitably linked to an amino group on a linker, for example by using CDAPconjugation. A further amino group in the linker for example ADH) may beconjugated to a carboxylic acid group on a protein, for example by usingcarbodiimide chemistry, for example by using EDAC. In an embodiment, thepneumococcal capsular saccharide(s) is conjugated to the linker firstbefore the linker is conjugated to the carrier protein. Alternativelythe linker may be conjugated to the carrier before conjugation to thesaccharide.

A combination of techniques may also be used, with somesaccharide-protein conjugates being prepared by CDAP, and some byreductive amination.

In general the following types of chemical groups on a protein carriercan be used for coupling/conjugation:

A) Carboxyl (for instance via aspartic acid or glutamic acid). In oneembodiment this group is linked to amino groups on polysaccharidesdirectly or to an amino group on a linker with carbodiimide chemistrye.g. with EDAC.

B) Amino group (for instance via lysine). In one embodiment this groupis linked to carboxyl groups on polysaccharides directly or to acarboxyl group on a linker with carbodiimide chemistry e.g. with EDAC.In another embodiment this group is linked to hydroxyl groups activatedwith CDAP or CNBr on polysaccharides directly or to such groups on alinker; to polysaccharides or linkers having an aldehyde group; topolysaccharides or linkers having a succinimide ester group.

C) Sulphydryl (for instance via cysteine). In one embodiment this groupis linked to a bromo or chloro acetylated polysaccharide or linker withmaleimide chemistry. In one embodiment this group is activated/modifiedwith bis diazobenzidine.

D) Hydroxyl group (for instance via tyrosine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.

E) Imidazolyl group (for instance via histidine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.

F) Guanidyl group (for instance via arginine).

G) Indolyl group (for instance via tryptophan).

On a saccharide, in general the following groups can be used for acoupling: OH, COOH or NH₂. Aldehyde groups can be generated afterdifferent treatments known in the art such as: periodate, acidhydrolysis, hydrogen peroxide, etc.

Direct Coupling Approaches:

Saccharide-OH+CNBr or CDAP----->cyanate ester+NH₂-Protein---->conjugate

Saccharide-aldehyde+NH₂-Protein---->Schiff base+NaCNBH3---->conjugate

Saccharide-COOH+NH₂-Protein+EDAC---->conjugate

Saccharide-NH₂+COOH-Protein+EDAC---->conjugate

Indirect Coupling Via Spacer (Linker) Approaches:

Saccharide-OH+CNBr or CDAP--->cyanateester+NH₂----NH₂---->saccharide----NH₂+COOH-Protein+EDAC>conjugate

Saccharide-OH+CNBr or CDAP---->cyanateester+NH₂-----SH----->saccharide----SH+SH-Protein (native Protein withan exposed cysteine or obtained after modification of amino groups ofthe protein by SPDP for instance)>saccharide-S—S-Protein

Saccharide-OH+CNBr or CDAP--->cyanateester+NH₂----SH------>saccharide----SH +maleimide-Protein (modificationof amino groups)---->conjugate

Saccharide-OH+CNBr or CDAP--->cyanateester+NH₂-----SH--->Saccharide-SH+haloacetylated-Protein---->Conjugate

Saccharide-COOH+EDAC+NH₂-----NH₂--->saccharideNH₂+EDAC+COOH-Protein---->conjugate

Saccharide-COOH+EDAC+NH₂----SH>saccharide----SH+SH-Protein (nativeProtein with an exposed cysteine or obtained after modification of aminogroups of the protein by SPDP for instance)----->saccharide-S—S-Protein

Saccharide-COOH+EDAC+NH₂---SH>saccharide----SH+maleimide-Protein(modification of amino groups)---->conjugate

Saccharide-COOH+EDAC+NH₂----SH--->Saccharide-SH+haloacetylated-Protein---->Conjugate

Saccharide-Aldehyde+NH₂-----NH₂---->saccharide---NH₂+EDAC+COOH-Protein---->conjugate

Note: instead of EDAC above, any suitable carbodiimide may be used.

In summary, the types of protein carrier chemical group that may begenerally used for coupling with a polysaccharide are amino groups (forinstance on lysine residues), COOH groups (for instance on aspartic andglutamic acid residues) and SH groups (if accessible) (for instance oncysteine residues).

In an embodiment, the Streptococcus pneumoniae serotype 6A capsularpolysaccharide of the invention is conjugated to the carrier protein(e.g. CRM-197) using CDAP chemistry. In one aspect, the CDAP chemistryuses a CDAP:PS6A ratio between 1:2 to 3:1, 1:1.5 to 2:1, e.g. 1:1. Inanother aspect, the CDAP conjugation is carried out using a couplingtime of between 50-130 minutes, 60-130 minutes, or 110-130 minutes. Thusthe present invention also provides, a process for preparing aStreptococcus pneumoniae serotype 6A capsular polysaccharide conjugate(e.g. a sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide of the invention) comprising conjugating a sizedStreptococcus pneumoniae serotype 6A capsular polysaccharide to acarrier protein (e.g. CRM-197) or to a linker (e.g. ADH) using CDAPchemistry using a CDAP:PS (polysaccharide) ratio between 1:2 to 3:1,1:1.5 to 2:1, e.g. 1:1.

In another aspect, the present invention provides, a process forpreparing a Streptococcus pneumoniae serotype 6A capsular polysaccharideconjugate (e.g. a sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide of the invention) comprising conjugating a sizedStreptococcus pneumoniae serotype 6A capsular polysaccharide to acarrier protein (e.g. CRM-197) using CDAP chemistry using a CDAP:PS(polysaccharide) ratio between 1:2 to 3:1, 1:1.5 to 2:1, e.g. 1:1. Inone aspect, the present invention also provides, a process for preparinga Streptococcus pneumoniae serotype 6A capsular polysaccharide conjugate(e.g. a sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide of the invention, such as a Streptococcus pneumoniaeserotype 6A capsular polysaccharide having an average size (e.g. M_(w))between 100-1000, 110-750, 150-500, 180-600, 210-490, 210-450, 180-400,210-400, 210-370, 220-360, 230-350, 240-340, 240-320, 240-310 or 250-310kDa) comprising conjugating a sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide to a carrier protein (e.g. CRM-197) or to alinker (e.g. ADH) using CDAP chemistry using a coupling time of between50-130 minutes, 60-130 minutes, or 110-130 minutes.

In another aspect, the present invention provides, a process forpreparing a Streptococcus pneumoniae serotype 6A capsular polysaccharideconjugate (e.g. a sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide of the invention, such as a Streptococcus pneumoniaeserotype 6A capsular polysaccharide having an average size (e.g. M_(w))between 100-1000, 110-750, 150-500, 180-600, 210-490, 210-450, 180-400,210-400, 210-370, 220-360, 230-350, 240-340, 240-320, 240-310 or 250-310kDa) comprising conjugating a sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide to a carrier protein (e.g. CRM-197) using CDAPchemistry using a coupling time of between 50-130 minutes, 60-130minutes, or 110-130 minutes. In one aspect, the pH for activation andcoupling is between pH 8 to pH9, suitably pH9.5. In another aspect, theconjugation is carried out in the presence of NaCl. For example, in0.1-3M NaCl, 0.1-2.5M NaCl, 1.5-2.5M NaCl or 2M NaCl.

The present invention also provides, a process for preparing aStreptococcus pneumoniae serotype 6A capsular polysaccharide conjugatecomprising (a) conjugation of a Streptococcus pneumoniae serotype 6Acapsular polysaccharide (e.g. a sized Streptococcus pneumoniae serotype6A capsular polysaccharide of the invention) to a carrier protein (e.g.CRM-197) and (b) diafiltration against a solution having a concentrationof NaCl below 150 mM (e.g. below 100 mM NaCl, below 50 mM NaCl, below 10mM NaCl) or using water (e.g. WFI, water for injection).

In another aspect, the present invention provides, a solution comprising6A-CRM197 in less than 150 mM NaCl. For example, less than 100 mM NaCl,less than 50 mM NaCl, less than 10 nM NaCl, or in the absence of sodiumchloride. In another aspect, the present invention provides, animmunogenic composition of the invention (e.g. 6A-CRM197) comprisingless than 150 mM NaCl. For example, less than 100 mM NaCl, less than 50mM NaCl, less than 10 mM NaCl, or in the absence of sodium chloride.

Ratio of Carrier Protein to Polysaccharide

In an embodiment, the ratio of carrier protein to S. pneumoniaepolysaccharide is between 1:5 and 5:1; e.g. between 1:0.5-4:1,1:1-3.5:1, 1.2:1-3:1, 1.5:1-2.5:1; e.g. between 1:2 and 2.5:1; 1:1 and2:1 (w/w; weight/weight). In an embodiment, the majority of theconjugates, for example 6, 7, 8, 9 or more of the conjugates have aratio of carrier protein to polysaccharide that is greater than 1:1, forexample 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1 or 1.6:1.

In an embodiment, the ratio of carrier protein to Streptococcuspneumoniae serotype 6A capsular polysaccharide in immunogeniccompositions of the invention is between 5:1 and 1:5, 4:1 and 1:1 or 2:1and 1:1, 1.5:1 and 1:1, 1.4:1 and 1.3:1 (for example 1.2:1, 1.5:1)(w/w).

The ratio of polysaccharide to carrier protein (w/w) in a conjugate maybe determined using the conjugate. The amount of protein is determinedusing a Lowry assay (for example Lowry et al. (1951) J. Biol. Chem. 193,265-275 or Peterson et al. Analytical Biochemistry 100, 201-220 (1979))and the amount of polysaccharide is determined using resorcinol assay(Monsigny et al. (1988) Anal. Biochem. 175, 525-530). The finalProtein/Polysaccharide ratio (w/w) on the sterilized conjugate isdetermined by the ratio of the Lowry/resorcinol concentrations.

Size of Capsular Polysaccharides in the Immunogenic Composition

Capsular polysaccharides of Streptococcus pneumoniae comprise repeatingoligosaccharide units which may contain up to 8 sugar residues. For areview of the oligosaccharide units for the key Streptococcus pneumoniaeserotypes see JONES, Christopher. Vaccines based on the cell surfacecarbohydrates of pathogenic bacteria. An. Acad. Bras. Ciênc., June 2005,vol. 77, no. 2, p. 293-324. Table II ISSN 0001-3765. In one embodiment,a capsular polysaccharide may be a full length polysaccharide, howeverin others it may be a shorter than native length polysaccharide chain ofrepeating units. In one embodiment, the Streptococcus pneumoniaeserotype capsular polysaccharide conjugates post conjugation should bereadily filterable through a 0.2 micron filter such that a yield of morethan 95% is obtained post filtration compared with the pre filtrationsample.

In addition to the sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide conjugate of the invention, the immunogenic compositionof the invention may comprise one or more (poly)saccharide conjugatesfrom Streptococcus pneumoniae serotypes other than 6A (e.g. 6B and/or23F) wherein the average size (e.g. weight-average molecular weight;M_(w)) of the (poly)saccharide before conjugation is above 80 kDa, 100kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 700 kDa or 1000 kDa. Forexample, the immunogenic composition of the invention may comprise oneor more (poly)saccharide conjugates from Streptococcus pneumoniaeserotypes other than 6A wherein the average size (e.g. weight-averagemolecular weight; M_(w)) of the (poly)saccharide before conjugation isbetween 80-100 kDa, 100-200 kDa, 200-300 kDa, 300-400 kDa, 400-500 kDa,500-1000 kDa or 1000-1400 kDa. In one embodiment, the immunogeniccomposition comprises (i) sized a Streptococcus pneumoniae serotype 6Acapsular polysaccharide conjugate and (ii) one or more (poly)saccharideconjugates with an average size of saccharide before conjugation of50-1600, 80-1400, 100-1000, 150-500, or 200-400 kDa (note that whereaverage size is M_(w), ‘kDa’ units should be replaced herein with‘×10³’).

In an embodiment, the immunogenic composition of the invention comprisesa serotype 1 S. pneumoniae polysaccharide having an average size (M_(w))of between 100-1000, 200-800, 250-600, or 300-400 kDa. In an embodiment,the immunogenic composition of the invention comprises a serotype 4 S.pneumoniae polysaccharide having an average size (M_(w)) of between50-500, 60-300, 70-150, or 75-125 kDa. In an embodiment, the immunogeniccomposition of the invention comprises a serotype 5 S. pneumoniaepolysaccharide having an average size (M_(w)) of between 100-1000,100-700, 100-350, or 150-300 kDa. In an embodiment, the immunogeniccomposition of the invention comprises a serotype 6B S. pneumoniaepolysaccharide having an average size (M_(w)) of between 200-1800,500-1800, 600-1800, 900-1660, or 1000-1400 kDa. In an embodiment, theimmunogenic composition of the invention comprises a serotype 7F S.pneumoniae polysaccharide having an average size (M_(w)) of between50-1000, 100-750, 150-500, or 200-300 kDa. In an embodiment, theimmunogenic composition of the invention comprises a serotype 9V S.pneumoniae polysaccharide having an average size (M_(w)) of between50-1000, 100-750, 150-500, 200-400, or 250-300 kDa. In an embodiment,the immunogenic composition of the invention comprises a serotype 14 S.pneumoniae polysaccharide having an average size (M_(w)) of between50-1000, 100-750, 150-500, or 200-250 kDa. In an embodiment, theimmunogenic composition of the invention comprises a 18C S. pneumoniaepolysaccharide having an average size (M_(w)) of between 50-1000,50-750, 50-500, 50-190, 50-150 or 80-110 kDa. In an embodiment, theimmunogenic composition of the invention comprises a 19A S. pneumoniaepolysaccharide having an average size (M_(w)) of between 50-800,110-700, 110-300, 120-200, 130-180, 140-160 or 80-130 kDa. In anembodiment, the immunogenic composition of the invention comprises aserotype 19F S. pneumoniae polysaccharide having an average size (M_(w))of between 50-1000, 100-750, 100-500, 100-190 or 120-180 kDa. In anembodiment, the immunogenic composition of the invention comprises aserotype 23F S. pneumoniae polysaccharide having an average size (M_(w))of between 500-1500, 600-1500, 700-1300, 900-1250, 800-1100, or 900-1000kDa. In an embodiment, the immunogenic composition of the inventioncomprises a serotype 22F S. pneumoniae polysaccharide having an averagesize (M_(w)) of between 50-800, 110-700, 110-300, 120-200, 130-180,150-170, 100-190, 100-150, 95-125 or 100-115 kDa.

In an embodiment, the immunogenic composition of the invention comprises1 or more, native capsular polysaccharides from different S. pneumoniaeserotypes. In another embodiment, the immunogenic composition comprisesStreptococcus pneumoniae polysaccharides from at least 10 serotypesconjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7,8, or 9 of the S. pneumoniae polysaccharide serotypes is nativepolysaccharide. In another embodiment, the immunogenic composition ofthe invention comprises native Streptococcus pneumoniae capsularserotype 6B polysaccharide. In another embodiment, the immunogeniccomposition of the invention comprises native Streptococcus pneumoniaecapsular serotype 23F polysaccharide.

In an aspect of the invention, the immunogenic composition comprisesStreptococcus pneumoniae polysaccharides from at least 10 serotypesconjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7,8, or 9 of the S. pneumoniae polysaccharide serotypes is sized by afactor up to ×2, ×3, ×4, ×5, ×6, ×7, ×8, ×9 or ×10. In one embodiment ofthis aspect, the majority of the polysaccharides, for example 6, 7, 8 ormore of the polysaccharide serotypes are sized by a factor up to ×2, ×3,×4, ×5, ×6, ×7, ×8, ×9 or ×10. For example, sizing may be from a factorof between ×2 to ×6, ×2 to ×5, ×2 to ×4, or ×3 to ×6, ×3 to ×5 or ×3 to×4.

In an embodiment, the majority of S. pneumoniae polysaccharides in theimmunogenic composition are sized. In an embodiment, the majority of S.pneumoniae polysaccharide serotypes in the immunogenic composition aresized. In one aspect, S. pneumoniae polysaccharides in the immunogeniccomposition are sized mechanical cleavage, for instance bymicrofluidisation or sonication. In another aspect, S. pneumoniaepolysaccharides in the immunogenic composition are sized by chemicalcleavage, e.g. treatment with acetic acid or periodate. Sizing is by afactor of no more than ×20, ×10, ×8, ×6, ×5, ×4, ×3 or ×2.

In an embodiment, the immunogenic composition comprises S. pneumoniaeconjugates that are a mixture of native polysaccharides andpolysaccharides that are sized by a factor of no more than ×20, ×10, ×8,×6, ×5, ×4, ×3 or ×2. In one aspect of this embodiment, the majority ofthe polysaccharides, for example 6, 7, 8, 9, 10 or more of thepolysaccharides are sized by a factor of up to ×2, ×3, ×4, ×5 or ×6.

Dosage

In general, the immunogenic composition of the invention may comprise adose of each saccharide conjugate between 0.1 and 20 μg, 1 and 10 μg or1 and 3 μg of saccharide.

In an embodiment, in the immunogenic composition of the presentinvention the dose of the Streptococcus pneumoniae 6A polysaccharideconjugate is between 1 and 10 μg, 1 and 5 μg, or 1 and 3 μg ofsaccharide (e.g. 2 μg).

In an embodiment, the immunogenic composition of the invention containseach S. pneumoniae capsular saccharide at a dose of between 0.1-20 μg;0.5-10 μg; 0.5-5 μg or 1-3 μg of saccharide. In an embodiment, capsularpolysaccharides may be present at different dosages, for example somecapsular polysaccharides may be present at a dose of around or exactly 1μg or some capsular polysaccharides may be present at a dose of aroundor exactly 3 μg. In an embodiment, polysaccharides from serotypes 3, 18Cand 19F are present at a higher dose than other polysaccharides. In anembodiment, polysaccharides from serotypes 4, 18C and 19F are present ata higher dose than other polysaccharides. In one aspect of thisembodiment, serotypes 3, 18C and 19F are present at a dose of around orexactly 3 μg whilst other polysaccharides in the immunogenic compositionare present at a dose of around or exactly 1 μg. In one aspect of thisembodiment, serotypes 4, 18C and 19F are present at a dose of around orexactly 3 μg whilst other polysaccharides in the immunogenic compositionare present at a dose of around or exactly 1μg.

“Around” or “approximately” are defined as within 10% more or less ofthe given figure for the purposes of the invention.

Streptococcus pneumoniae Proteins

The immunogenic composition of the invention may also compriseStreptococcus pneumoniae proteins, herein termed Streptococcuspneumoniae proteins of the invention. Such proteins may be used ascarrier proteins, or may be present as free proteins, or may be presentboth as carrier proteins and as free proteins. In an embodiment, theimmunogenic composition of the invention further comprises one or moreunconjugated or conjugated S. pneumoniae proteins. In an embodiment, theimmunogenic composition of the invention further comprises one or moreunconjugated S. pneumoniae proteins. For example, immunogeniccompositions of the invention may comprise unconjugated pneumolysin,e.g. dPly, and unconjugated pneumococcal PhtD.

The Streptococcus pneumoniae proteins of the invention are eithersurface exposed, at least during part of the life cycle of thepneumococcus, or are proteins which are secreted or released by thepneumococcus. In an embodiment, the proteins of the invention areselected from the following categories, such as proteins having a TypeII Signal sequence motif of LXXC (where X is any amino acid, e.g., thepolyhistidine triad family (PhtX)), choline binding proteins (e.g. CbpX,PcpA), proteins having a Type I Signal sequence motif (e.g., Sp101),proteins having a LPXTG motif (where X is any amino acid, e.g., Sp128,Sp130), and toxins (e.g., Ply). Preferred examples within thesecategories (or motifs) are the following proteins, or immunologicallyfunctional equivalents thereof. Thus, the immunogenic composition of theinvention may comprise one or more S. pneumoniae proteins selected fromPoly Histidine Triad family (PhtX), Choline Binding Protein family(CbpX), CbpX truncates, pneumococcal autolysin LytX family (LytA(N-acetylmuramoyl-l-alanine amidase), LytB, LytC), LytX truncates, CbpXtruncate-LytX truncate chimeric proteins, detoxified pneumolysin (Ply),PspA, PsaA, Sp128, Sp101, Sp130, Sp125 and Sp133. In a furtherembodiment, the immunogenic composition of the invention comprises 2 ormore proteins selected from the group consisting of the Poly HistidineTriad family (PhtX), Choline Binding Protein family (CbpX), CbpXtruncates, LytX family, LytX truncates, CbpXtruncate-LytXtruncatechimeric proteins (or fusions), pneumolysin (Ply), PspA, PsaA, andSp128. In a further embodiment, the immunogenic composition comprises 2or more proteins selected from the group consisting of the PolyHistidine Triad family (PhtX), Choline Binding Protein family (CbpX),CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytX truncatechimeric proteins (or fusions), pneumolysin (Ply), and Sp128.

The Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB,PhtD, and PhtE. The family is characterized by a lipidation sequence,two domains separated by a proline-rich region and several histidinetriads, possibly involved in metal or nucleoside binding or enzymaticactivity, (3-5) coiled-coil regions, a conserved N-terminus and aheterogeneous C terminus. It is present in all strains of pneumococcitested. Homologous proteins have also been found in other Streptococciand Neisseria. In one embodiment of the invention, the immunogeniccomposition comprises PhtD. It is understood, however, that the termsPht A, B, D, and E refer to proteins having sequences disclosed in thecitations below as well as variants thereof that have a sequencehomology that is at least 90% identical to the proteins described below,e.g. amino acids 21-838 of SEQ ID NO: 4 of WO00/37105. In an embodimentit is at least 95% identical and in another embodiment it is 97%identical.

With regards to the PhtX proteins, PhtA is disclosed in WO 98/18930, andis also referred to Sp36. As noted above, it is a protein from thepolyhistidine triad family and has the type II signal motif of LXXC.PhtD is disclosed in WO 00/37105, and is also referred to Sp036D. Asnoted above, it also is a protein from the polyhistidine triad familyand has the type II LXXC signal motif. PhtB is disclosed in WO 00/37105,and is also referred to Sp036B. Another member of the PhtB family is theC3-Degrading Polypeptide, as disclosed in WO 00/17370. This protein alsois from the polyhistidine triad family and has the type II LXXC signalmotif. A preferred immunologically functional equivalent is the proteinSp42 disclosed in WO 98/18930. A PhtB truncate (approximately 79 kD) isdisclosed in WO99/15675 which is also considered a member of the PhtXfamily. PhtE is disclosed in WO00/30299 and is referred to as BVH-3.Where any Pht protein is referred to herein, it is meant thatimmunogenic fragments or fusions thereof of the Pht protein can be used.For example, a reference to PhtX includes immunogenic fragments orfusions thereof from any Pht protein.

In one embodiment, the S. pneumoniae protein selected from member(s) ofthe Polyhistidine Triad family is PhtD. The term “PhtD” as used hereinincludes the full length protein with the signal sequence attached orthe mature full length protein with the signal peptide (for example 20amino acids at N-terminus) removed, and immunogenic fragments, variantsand/or fusion proteins thereof, e.g. SEQ ID NO: 4 of WO00/37105. In oneaspect, PhtD is the full length protein with the signal sequenceattached e.g. SEQ ID NO: 4 of WO00/37105. In another aspect, PhtD is asequence comprising the mature full length protein with the signalpeptide (for example 20 amino acids at N-terminus) removed, e.g. aminoacids 21-838 of SEQ ID NO: 4 of WO00/37105. Suitably, the PhtD sequencecomprises an N-terminal methionine. The present invention also includesPhtD polypeptides which are immunogenic fragments of PhtD, variants ofPhtD and/or fusion proteins of PhtD. For example, as described inWO00/37105, WO00/39299, U.S. Pat. No. 6,699,703 and WO09/12588.

Where immunogenic fragments of PhtD proteins are used (separately or aspart of a fusion protein), these immunogenic fragments will be at leastabout 15, at least about 20, at least about 40, or at least about 60contiguous amino acid residues in length, e.g. from a PhtD amino acidsequence in WO00/37105 or WO00/39299, such as SEQ ID NO: 4 ofWO00/37105. In an embodiment of the invention, immunogenic fragments ofPhtD protein comprise at least about 15, at least about 20, at leastabout 40, or at least about 60 contiguous amino acid residues of thesequence shown in SEQ ID NO: 4 of WO00/37105, wherein said polypeptideis capable of eliciting an immune response specific for said amino acidsequence. In an embodiment, the immunogenic composition of the inventioncomprises an immunogenic fragment of PhtD, for example described inWO09/12601, WO01/98334 and WO09/12588. Where immunogenic fragments ofPhtD proteins are used (separately or as part of a fusion protein), eachimmunogenic fragment optionally contains one or more histidine triadmotif(s) of such polypeptides. A histidine triad motif is the portion ofpolypeptide that has the sequence HxxHxH where H is histidine and x isan amino acid other than histidine. In an embodiment of the presentinvention, the or each immunogenic fragment contains exactly or at least2, 3, 4 or 5 histidine triad motifs (optionally, with native PhtDsequence between the 2 or more triads, or intra-triad sequence) wherethe immunogenic fragment is more than 50, 60, 70, 80, 90 or 100%identical to a native pneumococcal intra-triad PhtD sequence (e.g. theintra-triad sequence shown in SEQ ID NO: 4 of WO00/37105). Immunogenicfragments of PhtD proteins optionally contain one or more coiled coilregions of such polypeptides. A coiled coil region is a region predictedby “Coils” algorithm Lupus, A et al (1991) Science 252; 1162-1164. In anembodiment of the present invention, each immunogenic fragment containsexactly or at least 2, 3 or 4 coiled coil regions. In an embodiment ofthe present invention, the or each immunogenic fragment contains exactlyor at least 2, 3 or 4 coiled coil regions where the immunogenic fragmentis more than 50, 60, 70, 80, 90, 95, 96 or 100% identical to a nativepneumococcal PhtD sequence (e.g. the sequence shown in SEQ ID NO: 4 ofWO00/37105). In another embodiment of the present invention, theimmunogenic fragment includes one or more histidine triad motif as wellas at least 1, 2, 3 or 4 coiled coil regions.

In the case where the PhtD polypeptide is a variant, the variation isgenerally in a portion thereof other than the histidine triad residuesand the coiled-coil region, although variations in one or more of theseregions may be made. In accordance with the present invention, apolypeptide variant includes sequences in which one or more amino acidsare substituted and/or deleted and/or inserted compared to the wild typesequence. Amino acid substitution may be conservative ornon-conservative. In one aspect, amino acid substitution isconservative. Substitutions, deletions, insertions or any combinationthereof may be combined in a single variant so long as the variant is animmunogenic polypeptide. Variants of PhtD typically include anyimmunogenic fragment or variation of PhtD which shares at least 80, 90,95, 96, 98, or 99% amino acid sequence identity with a wild-type PhtDsequence, e.g. SEQ ID NO: 4 of WO00/37105. In an embodiment, the presentinvention includes immunogenic fragments and/or variants in whichseveral, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acid(s) aresubstituted, deleted, or added in any combination. In anotherembodiment, the present invention includes immunogenic fragments and/orvariants which comprise a B-cell or T-cell epitope. Such epitopes may bepredicted using a combination of 2D-structure prediction, e.g. using thePSIPRED program (from David Jones, Brunel Bioinformatics Group, Dept.Biological Sciences, Brunel University, Uxbridge UB8 3PH, UK) andantigenic index calculated on the basis of the method described byJameson and Wolf (CABIOS 4:181-186 [1988]).

In an embodiment of the invention, PhtD and its immunogenic fragments,variants and/or fusion proteins thereof comprise an amino acid sequencesharing at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity withamino acid sequence 21 to 838 of SEQ ID NO:4 of WO00/37105. In anotherembodiment of the invention, PhtD and its immunogenic fragments,variants and/or fusion proteins thereof have an amino acid sequencesharing at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity withamino acid sequence 21 to 838 of SEQ ID NO:4 of WO00/37105. Suitably,PhtD and its immunogenic fragments, variants and/or fusion proteinsthereof comprise an amino acid sequence having an N-terminal methionine.In another embodiment of the invention, PhtD and its immunogenicfragments, variants and/or fusion proteins thereof comprise at leastabout 15, at least about 20, at least about 40, or at least about 60 orat least about 100, or at least about 200, or at least about 400 or atleast about 800 contiguous amino acid residues of the sequence shown inSEQ ID NO: 4 of WO00/37105.

Pneumolysin (Ply) is a multifunctional toxin witha distinct cytolytic(hemolytic) and complement activation activities (Rubins et al., Am.Respi. Cit Care Med, 153:1339-1346 (1996)). The toxin is not secreted bypneumococci, but it is released upon lysis of pneumococci under theinfluence of autolysin. Its effects include e.g., the stimulation of theproduction of inflammatory cytokines by human monocytes, the inhibitionof the beating of cilia on human respiratory epithelial, the decrease ofbactericidal activity and migration of neutrophils, and in the lysis ofred blood cells, which involves binding to cholesterol. Because it is atoxin, it needs to be detoxified (i.e., non-toxic to a human whenprovided at a dosage suitable for protection) before it can beadministered in vivo. Expression and cloning of wild-type or nativepneumolysin is known in the art. See, for example, Walker et al. (InfectImmun, 55:1184-1189 (1987)), Mitchell et al. (Biochim Biophys Acta,1007:67-72 (1989) and Mitchell et al (NAR, 18:4010 (1990)).Detoxification of Ply can be conducted by chemical means, e.g., subjectto formalin or glutaraldehyde treatment or a combination of both (WO04081515, PCT/EP2005/010258). Such methods are known in the art forvarious toxins. Alternatively, Ply can be genetically detoxified. Thus,the invention encompasses derivatives of pneumococcal proteins which maybe, for example, mutated proteins. The term “mutated” is used herein tomean a molecule which has undergone deletion, addition or substitutionof one or more amino acids using known techniques for site directedmutagenesis or any other conventional method. For example, as describedabove, a mutant Ply protein may be altered so that it is biologicallyinactive whilst still maintaining its immunogenic epitopes, see, forexample, WO90/06951, Berry et al. (Infect Immun, 67:981-985 (1999)) andWO99/03884.

As used herein, it is understood that the term “Ply” encompasses mutatedpneumolysin and detoxified pneumolysin (dPly) suitable for medical use(i.e., non toxic). Concerning the Choline Binding Protein family (CbpX),members of that family were originally identified as pneumococcalproteins that could be purified by choline-affinity chromatography. Allof the choline-binding proteins are non-covalently bound tophosphorylcholine moieties of cell wall teichoic acid andmembrane-associated lipoteichoic acid. Structurally, they have severalregions in common over the entire family, although the exact nature ofthe proteins (amino acid sequence, length, etc.) can vary. In general,choline binding proteins comprise an N terminal region (N), conservedrepeat regions, a proline rich region (P) and a conserved cholinebinding region (C), made up of multiple repeats, that comprisesapproximately one half of the protein. As used in this application, theterm “Choline Binding Protein family (CbpX)” is selected from the groupconsisting of Choline Binding Proteins as identified in WO97/41151,Choline binding protein A, CbpA (also referred to as PbcA (C3-bindingprotein A), SpsA (Streptococcus pneumoniae secretory IgA bindingprotein), PspC (pneumococcal surface protein C)), Choline bindingprotein D (CbpD), and Choline binding protein G (CbpG). CbpA isdisclosed in WO97/41151. CbpD and CbpG are disclosed in WO00/29434. PspCis disclosed in WO97/09994. PbcA is disclosed in WO98/21337. SpsA is aCholine binding protein disclosed in WO 98/39450. In an embodiment, theCholine Binding Proteins is CbpA. Another Choline Binding Protein ispneumococcal choline-binding protein A (PcpA) (Sanchez-Beato et al FEMSMicrobiology Letters 164 (1998) 207-214).

Another preferred embodiment is CbpX truncates wherein “CbpX” is CbpA,CbpD or CbpG and “truncates” refers to CbpX proteins lacking 50% or moreof the Choline binding region (C). Another preferred embodiment is PcpAtruncates wherein “truncates” refers to PcpA proteins lacking 50% ormore of the Choline binding region (C). In an embodiment, CbpX truncatesor PcpA truncates lack the entire choline binding region. In anotherembodiment, the CbpX truncates or PcpA truncates lack (i) the cholinebinding region and (ii) a portion of the N-terminal half of the proteinas well, yet retain at least one repeat region. In another embodiment,the truncate has at least 2 repeat regions. Examples of such preferredembodiments are illustrated in WO99/51266 or WO99/51188, however, othercholine binding proteins lacking a similar choline binding region arealso contemplated within the scope of this invention.

The LytX family is membrane associated proteins associated with celllysis. The N-terminal domain comprises choline binding domain(s),however the LytX family does not have all the features found in the CbpAfamily noted above and thus for the present invention, the LytX familyis considered distinct from the CbpX family. In contrast with the CbpXfamily, the C-terminal domain contains the catalytic domain of the LytXprotein family. The family comprises LytA, LytB and LytC. With regardsto the LytX family, LytA is disclosed in Ronda et al., Eur J Biochem,164:621-624 (1987). LytB is disclosed in WO 98/18930, and is alsoreferred to as Sp46. LytC is also disclosed in WO 98/18930, and is alsoreferred to as Sp91. A preferred member of that family is LytC.

Another preferred embodiment are LytX truncates wherein “LytX” is LytA,LytB or LytC and “truncates” refers to LytX proteins lacking 50% or moreof the Choline binding region. Suitably such proteins lack the entirecholine binding region. Yet another preferred embodiment of thisinvention are CbpX truncate-LytX truncate chimeric proteins (orfusions). In an embodiment, the CbpX truncate-LytX truncate chimericprotein comprises the repeat regions of CbpX and the C-terminal portion(Cterm, i.e., lacking the choline binding domains) of LytX (e.g.,LytCCterm or Sp91Cterm). In another embodiment, CbpX is selected fromthe group consisting of CbpA, PbcA, SpsA and PspC. In anotherembodiment, it is CbpA. In an embodiment, LytX is LytC (also referred toas Sp91). Another embodiment of the present invention is a PspA or PsaAtruncates lacking the choline binding domain (C) and expressed as afusion protein with LytX. In an embodiment, LytX is LytC.

PsaA and transmembrane deletion variants thereof have been described byBerry & Paton, Infect Immun 1996 December; 64(12):5255-62. PspA andtransmembrane deletion variants thereof have been disclosed in, forexample, U.S. Pat. No. 5,804,193, WO 92/14488, and WO 99/53940.

Sp128 and Sp130 are disclosed in WO00/76540. Sp125 is an example of apneumococcal surface protein with the Cell Wall Anchored motif of LPXTG(i.e. leucine-profine-X-threonine-glycine where X is any amino acid).Any protein within this class of pneumococcal surface protein with thismotif has been found to be useful within the context of this invention,and is therefore considered a further protein of the invention. Sp125itself is disclosed in WO 98/18930, and is also known as ZmpB—a zincmetalloproteinase. Sp101 is disclosed in WO 98/06734 (where it has thereference # y85993). It is characterized by a Type I signal sequence.Sp133 is disclosed in WO 98/06734 (where it has the reference # y85992).It is also characterized by a Type I signal sequence.

The proteins of the invention may also be beneficially combined. Bycombined is meant that the immunogenic composition comprises all of theproteins from within the following combinations, either as carrierproteins or as free proteins or a mixture of the two. For example, in acombination of two proteins as set out hereinafter, both proteins may beused as carrier proteins, or both proteins may be present as freeproteins, or both may be present as carrier and as free protein, or onemay be present as a carrier protein and a free protein whilst the otheris present only as a carrier protein or only as a free protein, or onemay be present as a carrier protein and the other as a free protein.Where a combination of three proteins is given, similar possibilitiesexist. Preferred combinations include, but are not limited to PhtD+CbpXrepeat regions, PhtD+Ply, PhtD+Sp128, PhtD+PsaA, PhtD+PspA, PhtA+CbpXrepeat regions, PhtA+CbpX repeat regions−Sp91Cterm chimeric or fusionproteins, PhtA+Ply, PhtA+Sp128, PhtA+PsaA, PhtA+PspA, CbpX repeatregions+LytC, CbpX repeat regions+PspA, CbpX repeat regions+PsaA, CbpXrepeat regions+Sp128, CbpX repeat regions+LytC, CbpX repeatregions+PspA, CbpX repeat regions+PsaA, CbpX repeat regions+Sp128, CbpXrepeat regions+PhtD, CbpX repeat regions+PhtA. In an embodiment, CbpXrepeat regionsis from CbpA. In another embodiment, it is from CbpA.Other combinations include 3 protein combinations such as PhtD+CbpXrepeat regions+Ply, and PhtA+CbpX repeat regions+PhtD. In oneembodiment, the immunogenic composition comprises detoxified pneumolysinand PhtD or PhtDE as carrier proteins. In a further embodiment, theimmunogenic composition comprises detoxified pneumolysin and PhtD orPhtDE as free proteins.

The total content of protein antigens in the vaccine will typically bein the range 1-100 μg, or 5-80 μg, e.g. in the range 50-70 μg. Forexample, in one aspect, the immunogenic composition of the inventioncomprises 26 μg-45 μg (for example 26 μg-40 μg, 28 μg-35 μg or around 30μg) of each S. pneumoniae protein, per human dose. In another aspect,the immunogenic composition comprises 26 μg-45 μg (for example 26 μg-40μg, 28 μg-35 μg or around 30 μg) of PhtD, per human dose. In anotheraspect, the immunogenic composition of the invention comprises 26 μg-45μg (for example 26 μg-40 μg, 28 μg-35 μg or around 30 μg) of pneumolysin(e.g. dPly), per human dose.

By the term “human dose” is meant a dose which is in a volume suitablefor human use. Generally this is between 0.25 and 1.5 ml. In oneembodiment, a human dose is 0.5 ml. In a further embodiment, a humandose is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In afurther embodiment, a human dose is between 1 ml and 1.5 ml. In anotherembodiment, in particular when the immunogenic composition is for thepaediatric population, a human dose may be less than 0.5 ml such asbetween 0.25 and 0.5 ml.

Adjuvants

The immunogenic compositions of the present invention may be adjuvanted,particularly when intended for use in an elderly population but also foruse in infant populations. Thus, a further aspect is an immunogeniccomposition of the invention which further comprises an adjuvant.Suitable adjuvants include an aluminum salt such as aluminum hydroxidegel or aluminum phosphate or alum, but a suitable adjuvant may also be asalt of calcium, magnesium, iron or zinc, or may be an insolublesuspension of acylated tyrosine, or acylated sugars, cationically oranionically derivatized polysaccharides, or polyphosphazenes. In oneaspect of the invention, the adjuvant is an aluminium salt, e.g.aluminium phosphate. In a further aspect, the adjuvant comprises (per0.5 mL dose) 100-750, 200-500, or 300-400 μg Al (aluminium) as aluminiumphosphate.

In one embodiment, the adjuvant is a preferential inducer of a Th1 typeof response. Such high levels of Th1-type cytokines tend to favour theinduction of cell mediated immune responses to a given antigen, whilsthigh levels of Th2-type cytokines tend to favour the induction ofhumoral immune responses to the antigen. Suitable adjuvant systems whichpromote a predominantly Th1 response include: Monophosphoryl lipid A ora derivative thereof, particularly 3-de-O-acylated monophosphoryl lipidA (3D-MPL) (for its preparation see GB 2220211 A); and a combination ofmonophosphoryl lipid A, suitably 3-de-O-acylated monophosphoryl lipid A,together with either an aluminum salt (for instance aluminum phosphateor aluminum hydroxide) or an oil-in-water emulsion. In suchcombinations, antigen and 3D-MPL are contained in the same particulatestructures, allowing for more efficient delivery of antigenic andimmunostimulatory signals. Studies have shown that 3D-MPL is able tofurther enhance the immunogenicity of an alum-adsorbed antigen [Thoelenet al. Vaccine (1998) 16:708-14; EP 689454-61].

The immunogenic composition may comprise a sized Streptococcuspneumoniae serotype 6A capsular polysaccharide (and optionally animmunostimulant) adsorbed onto a metal salt (such as an aluminium salt,for example Aluminium phosphate or Aluminium hydroxide). The term“immunostimulant” as used herein means a substance that stimulates animmune response, in particular an adjuvant which stimulates the immunesystem of a host animal (e.g. human) to which it is administered andthereby increases the protective effect produced by a antigenadministered to that animal, as compared to the effect which would beproduced by administration of the antigen alone. For aluminium basedvaccine formulations wherein the antigen is typically adsorbed ontoaluminium salt for one hour at room temperature under agitation.

Adjuvants Comprising Additional Immunostimulants

The adjuvant of the invention may comprise immunostimulants, such assaponins (e.g. QS21) and/or 3D-MPL. Examples of immunostimulants aredescribed herein and in “Vaccine Design—The Subunit and AdjuvantApproach” 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M.F., and Newman, M. J., Plenum Press, New York and London, ISBN0-306-44867-X. In one aspect of the present invention the adjuvantcomprises QS21, monophosphoryl lipid A (MPL), phospholipid and sterol,presented in the form of a liposome.

QS-21 is a purified saponin fraction from the bark extracts of the SouthAmerican tree Quillaja saponaria. QS21 typically comprises two principalisomers that share a triterpene, a branched trisaccharide, and aglycosylated pseudodimeric acyl chain. The two isomeric forms differ inthe constitution of the terminal sugar within the linear tetrasaccharidesegment, wherein the major isomer, QS-21-Api incorporates a β-D-apioseresidue, and the minor isomer, QS-21-Xyl terminates in a β-D-xylosesubstituent. (Cleland, J. L. et al. J. Pharm. Sci. 1996, 85, 22-28).QS21 may be prepared by HPLC purification from Quil A™. Quil A™ wasdescribed as having adjuvant activity by Dalsgaard et al. in 1974(“Saponin adjuvants”, Archiv. für die gesamte Virusforschung, Vol. 44,Springer Verlag, Berlin, p 243-254). Methods for production of QS21 aredescribed in U.S. Pat. No. 5,057,540 (where QS21 is described as QA21)and EP0362278. In an embodiment, immunogenic compositions of theinvention contain QS21 in substantially pure form, that is to say, theQS21 comprises at least 90%, for example at least 95%, or at least 98%of the immunogenic composition (i.e. the QS21 composition contains atleast 90%, for example at least 95%, or at least 98% QS21). The dose ofQS21 is suitably able to enhance an immune response to an antigen in ahuman. In particular a suitable QS21 amount is that which improves theimmunological potential of the composition compared to the unadjuvantedcomposition, or compared to the composition adjuvanted with another QS21amount, whilst being acceptable from a reactogenicity profile. QS21 canbe used, for example, at an amount of 1 to 100 μg per composition dose,for example in an amount of 10 to 50 μg per composition dose.

Monophosphoryl lipid A (MPL) is a nontoxic derivative of thelipopolysaccharide (LPS) of gram-negative bacteria, e.g. Salmonellaminnesota R595. It retains adjuvant properties of the LPS whiledemonstrating a reduced toxicity (Johnson et al. 1987 Rev. Infect. Dis.9 Suppl:S512-S516). MPL is composed of a series of 4′-monophosphoryllipid A species that vary in the extent and position of fatty acidsubstitution. It may be prepared by treating LPS with mild acid and basehydrolysis followed by purification of the modified LPS. For example,LPS may be refluxed in mineral acid solutions of moderate strength (e.g.0.1 M HCl) for a period of approximately 30 minutes. This processresults in dephosphorylation at the 1 position, and decarbohydration atthe 6′ position. The term “monophosphoryl lipid A (MPL)” as used hereinincludes derivatives of monophosphoryl lipid A. Derivatives ofmonophosphoryl lipid A include 3D-MPL and synthetic derivatives.

3D-MPL is 3-O-deacylated monophosphoryl lipid A (or 3 De-O-acylatedmonophosphoryl lipid A). Chemically it is a mixture of 3- deacylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. 3D-MPL isavailable under the trademark MPL® by GlaxoSmithKline Biologicals NorthAmerica. 3-O-deacylated monophosphoryl lipid A (3D-MPL). It has afurther reduced toxicity while again maintaining adjuvanticity, and maytypically be prepared by mild alkaline hydrolysis, see for example U.S.Pat. No. 4,912,094. Alkaline hydrolysis is typically performed inorganic solvent, such as a mixture of chloroform/methanol, by saturationwith an aqueous solution of weak base, such as 0.5 M sodium carbonate atpH 10.5. For further information on the preparation of 3D-MPL seeGB2220211A and WO02078637 (Corixa Corporation). In one aspect of thepresent invention small particle 3 D-MPL may be used. Small particle3D-MPL has a particle size such that it may be sterile-filtered througha 0.22 μm filter. Such preparations are described in InternationalPatent Application No. WO94/21292. In an embodiment, immunogeniccompositions of the invention comprise 3-O-Deacylated monophosphoryllipid A (3D-MPL).

The dose of monophosphoryl lipid A (MPL), e.g. 3D-MPL, is suitably ableto enhance an immune response to an antigen in a human. In particular asuitable monophosphoryl lipid A (MPL), e.g. 3D-MPL, amount is that whichimproves the immunological potential of the composition compared to theunadjuvanted composition, or compared to the composition adjuvanted withanother MPL amount, whilst being acceptable from a reactogenicityprofile. Monophosphoryl lipid A (MPL), e.g. 3D-MPL, can be used, forexample, at an amount of 1 to 100 μg per composition dose, for examplein an amount of 10 to 50 μg per composition dose.

Liposomes may be made from phospholipids (such as dioleoyl phosphatidylcholine, DOPC) and sterol, e.g. cholesterol, using techniques known inthe art. Such liposome carriers may carry the QS21 and/or monophosphoryllipid A (MPL), e.g. 3D-MPL. Suitable compositions of the invention arethose wherein liposomes are initially prepared without MPL (as describedin WO96/33739), and MPL is then added, suitably as small particles ofbelow 100 nm particles or particles that are susceptible to sterilefiltration through a 0.22 μm membrane. The MPL is therefore notcontained within the vesicle membrane (known as MPL out). Compositionswhere the MPL is contained within the vesicle membrane (known as MPL in)also form an aspect of the invention. The unconjugated S. pneumoniaeproteins can be contained within the vesicle membrane or containedoutside the vesicle membrane. Suitably soluble antigens are outside andhydrophobic or lipidated antigens are either contained inside or outsidethe membrane. Encapsulation within liposomes is described in U.S. Pat.No. 4,235,877.

The liposomes of the present invention may comprise a phospholipid, forexample a phosphatidylcholine, which may be non-crystalline at roomtemperature, for example egg yolk phosphatidylcholine, dioleoylphosphatidylcholine or dilauryl phosphatidylcholine. Suitably, thephospholipid is dioleoylphosphatidylcholine (DOPC). A further aspect isan immunogenic composition of the invention comprising 0.1 to 10 mg, 0.2to 7, 0.3 to 5, 0.4 to 2, or 0.5 to 1 mg (e.g. 0.4 to 0.6, 0.9 to 1.1,0.5 or 1 mg) phospholipid.

The liposomes of the present invention may comprise a sterol. The sterolincreases the stability of the liposome structure. Suitable sterolsinclude β-sitosterol, stigmasterol, ergosterol, ergocalciferol andcholesterol. These sterols are described in the art, for examplecholesterol is disclosed in the Merck Index, 11th Edn., page 341, as anaturally occurring sterol found in animal fat. In one particularembodiment of the invention, the sterol is cholesterol. Typically, thesterol may be added during formulation of the antigen preparation usingQS21 quenched with the sterol as described in WO96/33739. In anembodiment, the immunogenic compositions of the invention comprise 0.025to 2.5, 0.05 to 1.5, 0.075 to 0.75, 0.1 to 0.3, or 0.125 to 0.25 mg(e.g. 0.2 to 0.3, 0.1 to 0.15, 0.25 or 0.125 mg) sterol.

In one embodiment the the adjuvant comprises (per 0.5 mL dose) 0.1-10mg, 0.2-7, 0.3-5, 0.4-2, or 0.5-1 mg (e.g. 0.4-0.6, 0.9-1.1, 0.5 or 1mg) phospholipid (for instance DOPC), 0.025-2.5, 0.05-1.5, 0.075-0.75,0.1-0.3, or 0.125-0.25 mg (e.g. 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg)sterol (for instance cholesterol), 5-60, 10-50, or 20-30 μg (e.g. 5-15,40-50, 10, 20, 30, 40 or 50 μg) lipid A derivative (for instance3D-MPL), and 5-60, 10-50, or 20-30 μg (e.g. 5-15, 40-50, 10, 20, 30, 40or 50 μg) saponin (for instance QS21).

Liposomes of the invention will suitably be comprised in a liquidmedium. The liquid medium comprises physiologically acceptable liquidssuch as water, aqueous salt solutions and buffer solutions, e.g.phosphate buffered saline (PBS) etc. For example, immunogeniccompositions of the invention may comprise water and PBS.

In one aspect of the invention, the adjuvant is ASO1B (see e.g.WO96/33739). In another aspect of the invention, the adjuvant is AS01E(see e.g. WO2007/068907).

In some cases it may be advantageous that the immunogenic compositionsand vaccines of the present invention will further contain a stabiliser,for example other emulsifiers/surfactants, including caprylic acid(Merck index 10^(th) Edition, entry no. 1739), of which Tricaprylin isparticularly preferred.

Oil in Water Emulsion Adjuvants

Oil in water emulsion adjuvants per se have been suggested to be usefulas adjuvant compositions (EP 0 399 843B), also combinations of oil inwater emulsions and other active agents have been described as adjuvantsfor vaccines (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241). Otheroil emulsion adjuvants have been described, such as water in oilemulsions (U.S. Pat. No. 5,422,109; EP 0 480 982 B2) and water in oil inwater emulsions (U.S. Pat. No. 5,424,067; EP 0 480 981 B). All of whichform preferred oil emulsion systems (in particular when incorporatingtocols) that are suitable as adjuvants for use in compositions of thepresent invention.

A suitable oil emulsion (for example an oil-in-water emulsion) comprisesa metabolisible, non-toxic oil, such as squalane, a tocopherol such asalpha tocopherol and optionally an emulsifier (or surfactant) such aspolysorbate 80 (Tween™ 80). A sterol (for example cholesterol) may alsobe included. In one aspect of the invention, there is provided a vaccineor immunogenic composition comprising a sized S. pnemoniae serotype 6Acapsular polysaccharide and an adjuvant composition comprising an oil inwater emulsion, wherein the oil in water emulsion comprises 0.5-11 mgmetabolisable oil, (such as squalene), 0.5-12 mg tocol (such asalpha-tocopherol) and 0.4-5 mg emulsifying agent (such aspolyoxyethylene sorbitan monooleate), per human dose.

In order for any oil in water composition to be suitable for humanadministration, the oil phase of the emulsion system has to comprise ametabolisable oil. The meaning of the term metabolisable oil is known inthe art. Metabolisable can be defined as ‘being capable of beingtransformed by metabolism’ (Dorland's Illustrated Medical Dictionary,W.B. Sanders Company, 25th edition (1974)). The oil may be any vegetableoil, fish oil, animal oil or synthetic oil, which is not toxic to therecipient and is capable of being transformed by metabolism. Nuts,seeds, and grains are common sources of vegetable oils. Synthetic oilsare also part of this invention and can include commercially availableoils such as NEOBEE® and others. A particularly suitable metabolisableoil is squalene. Squalene(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is anunsaturated oil which is found in large quantities in shark-liver oil,and in lower quantities in olive oil, wheat germ oil, rice bran oil, andyeast, and is a particularly preferred oil for use in this invention.Squalene is a metabolisable oil, that it is an intermediate in thebiosynthesis of cholesterol (Merck index, 10th Edition, entry no. 8619).Suitably the metabolisable oil is present in the adjuvant composition inan amount of 0.5-10 mg, for example 1-10, 2-10, 3-9, 4-8, 5-7, or 5-6 mg(e.g. 2-3, 5-6, or 9-10 mg).

The oil in water emulsion suitably comprises a tocol. Tocols are knownin the art and are described in EP0382271. Suitably the tocol isalpha-tocopherol or a derivative thereof such as alpha-tocopherolsuccinate (also known as vitamin E succinate). Said tocol is suitablypresent in the adjuvant composition in an amount of 0.5-11 mg, forexample 1-11, 2-10, 3-9, 4-8, 5-7, 5-6 (e.g. 10-11, 5-6, 2.5-3.5 or 1-3mg). In a specific embodiment the tocol is present in an amount of 5.94mg or 2.38 mg. In a further embodiment, said tocol is suitably presentin the vaccine (or immunogenic) composition in an amount of 0.5-11 mg,for example 1-11, 2-10, 3-9, 4-8, 5-7, 5-6 (e.g. 10-11, 5-6, 2.5-3.5 or1-3 mg).

The oil in water emulsion may further comprise an emulsifying agent. Theemulsifying agent may suitably be polyoxyethylene sorbitan monooleate.In a particular embodiment the emulsifying agent may be selected fromthe group comprising: Polysorbate 80 (Tween™ 80). Said emulsifying agentis suitably present in the adjuvant composition in an amount of 0.1-5,0.2-5, 0.3-4, 0.4-3 or 2-3 mg (e.g. 0.4-1.2, 2-3 or 4-5 mg) emulsifyingagent.

The method of producing oil-in-water emulsions is known to the personskilled in the art. Commonly, the method comprises mixing thetocol-containing oil phase with a surfactant such as a PBS/Tween™80solution, followed by homogenisation using a homogenizer. A methodcomprising passing the mixture twice through a syringe needle would besuitable for homogenising small volumes of liquid. Equally, theemulsification process in microfluidiser (M110S Microfluidics machine,maximum of 50 passes, for a period of 2 minutes at maximum pressureinput of 6 bar (output pressure of about 850 bar)) could be adapted bythe man skilled in the art to produce smaller or larger volumes ofemulsion.

In an oil in water emulsion, the oil and emulsifier should be in anaqueous carrier. The aqueous carrier may be, for example, phosphatebuffered saline.

In an embodiment, the oil-in-water emulsion systems of the presentinvention have a small oil droplet size in the sub-micron range.Suitably the droplet sizes will be in the range 120 to 750 nm, or from120 to 600 nm in diameter. In an embodiment, the oil-in water emulsioncontains oil droplets of which at least 70% by intensity are less than500 nm in diameter, or at least 80% by intensity are less than 300 nm indiameter, or at least 90% by intensity are in the range of 120 to 200 nmin diameter.

The oil droplet size, i.e. diameter, according to the present inventionis given by intensity. There are several ways of determining thediameter of the oil droplet size by intensity. Intensity is measured byuse of a sizing instrument, suitably by dynamic light scattering such asthe Malvern Zetasizer 4000 or suitably the Malvern Zetasizer 3000HS. Afirst possibility is to determine the z average diameter (ZAD) bydynamic light scattering (PCS, Photon correlation spectroscopy); thismethod additionally gives the polydispersity index (PDI), and both theZAD and PDI are calculated with the cumulants algorithm. These values donot require the knowledge of the particle refractive index. A secondmean is to calculate the diameter of the oil droplet by determining thewhole particle size distribution by another algorithm, either theContin, or non-negative least squares (NNLS), or the automatic “Malvern”one (the default algorithm provided for by the sizing instrument). Mostof the time, as the particle refractive index of a complex compositionis unknown, only the intensity distribution is taken into consideration,and if necessary the intensity means originating from this distribution.

Lipopolysaccharide (LPS) or lipooligosaccharide (LOS) derivatives ormutations or lipid A derivatives described herein are designed to beless toxic (e.g. 3D-MPL) than native lipopolysaccharides

In one embodiment the adjuvant used for the compositions of theinvention comprises an oil in water emulsion made from a metabolisableoil (such as squalene), an emulsifier (such as Tween™ 80) and optionallya tocol (such as alpha tocopherol). In one embodiment the adjuvantcomprises (per 0.5 mL dose) 0.5-15, 1-13, 2-11, 4-8, or 5-6 mg (e.g.2-3, 5-6, or 10-11 mg) metabolisable oil (such as squalene), 0.1-10,0.3-8, 0.6-6, 0.9-5, 1-4, or 2-3 mg (e.g. 0.9-1.1, 2-3 or 4-5 mg)emulsifier (such as Tween™ 80) and optionally 0.5-20, 1-15, 2-12, 4-10,5-7 mg (e.g. 11-13, 5-6, or 2-3 mg) tocol (such as alpha tocopherol).

The adjuvant may optionally further comprise 5-60, 10-50, or 20-30 μg(e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 μg) lipid A derivative (forinstance 3D-MPL).

The adjuvant may optionally contain 0.025-2.5, 0.05-1.5, 0.075-0.75,0.1-0.3, or 0.125-0.25 mg (e.g. 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg)sterol (for instance cholesterol), 5-60, 10-50, or 20-30 μg (e.g. 5-15,40-50, 10, 20, 30, 40 or 50 μg) lipid A derivative (for instance3D-MPL), and 5-60, 10-50, or 20-30 μg (e.g. 5-15, 40-50, 10, 20, 30, 40or 50 μg) saponin (for instance QS21).

In one embodiment, the adjuvant used for the compositions of theinvention comprises aluminium phosphate and a lipid A derivative (suchas 3D-MPL). This adjuvant may comprise (per 0.5 mL dose) 100-750,200-500, or 300-400 μg Al as aluminium phosphate, and 5-60, 10-50, or20-30 μg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 μg) lipid A derivative(for instance 3D-MPL).

Method of Administration

The vaccine preparations containing immunogenic compositions of thepresent invention may be used to protect or treat a mammal susceptibleto infection, by means of administering said vaccine via a systemic ormucosal route. These administrations may include injection via theintramuscular (IM), intraperitoneal (IP), intradermal (ID) orsubcutaneous (SC) routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. Although the vaccineof the invention may be administered as a single dose, componentsthereof may also be co-administered together at the same time or atdifferent times (for instance pneumococcal saccharide conjugates couldbe administered separately, at the same time or 1-2 weeks after theadministration of the any bacterial protein component of the vaccine foroptimal coordination of the immune responses with respect to eachother). For co-administration, the optional Th1 adjuvant may be presentin any or all of the different administrations. In addition to a singleroute of administration, 2 different routes of administration may beused. For example, polysaccharide conjugates may be administered IM (orID) and bacterial proteins may be administered IN (or ID). In addition,the vaccines of the invention may be administered IM for priming dosesand IN for booster doses.

Following an initial vaccination, subjects may receive one or severalbooster immunizations adequately spaced.

Vaccine

The present invention further provides a vaccine containing theimmunogenic compositions of the invention and a pharmaceuticallyacceptable excipient or carrier.

Pharmaceutically acceptable excipients and carriers are well known andcan be selected by those of skill in the art. For example, thepharmaceutically acceptable excipient or carrier can include a buffer,such as Tris (trimethamine), phosphate (e.g. sodium phosphate), acetate,borate (e.g. sodium borate), citrate, glycine, histidine and succinate(e.g. sodium succinate), suitably sodium chloride, histidine, sodiumphosphate or sodium succinate. The pharmaceutically acceptable excipientmay include a salt, for example sodium chloride, potassium chloride ormagnesium chloride. Optionally, the pharmaceutically acceptableexcipient contains at least one component that stabilizes solubilityand/or stability. Examples of solubilizing/stabilizing agents includedetergents, for example, laurel sarcosine and/or tween (e.g. Tween 80).Examples of stabilizing agents also include poloxamer (e.g. poloxamer124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407). Thepharmaceutically acceptable excipient may include a non-ionicsurfactant, for example polyoxyethylene sorbitan fatty acid esters,Polysorbate-80 (Tween 80), Polysorbate-60 (Tween 60), Polysorbate-40(Tween 40) and Polysorbate-20 (Tween 20), or polyoxyethylene alkylethers (suitably polysorbate-80). Alternative solubilizing/stabilizingagents include arginine, and glass forming polyols (such as sucrose,trehalose and the like). The pharmaceutically excipient may be apreservative, for example phenol, 2-phenoxyethanol, or thiomersal. Otherpharmaceutically acceptable excipients include sugars (e.g. lactose,sucrose), and proteins (e.g. gelatine and albumin). Pharmaceuticallyacceptable carriers include water, saline solutions, aqueous dextroseand glycerol solutions. Numerous pharmaceutically acceptable excipientsand carriers are known in the art and are described, e.g., inRemington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 5th Edition (975).

According to a further aspect of the invention there is provided aprocess for making the immunogenic composition or vaccine of theinvention comprising the step of mixing S. pneumoniae capsularpolysaccharide (conjugates) of the invention, optionally with apharmaceutically acceptable excipient or carrier.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

The vaccines of the present invention may be stored in solution orlyophilized. In an embodiment, the solution is lyophilized in thepresence of a sugar such as sucrose or lactose. It is still furtherpreferable that they are lyophilized and extemporaneously reconstitutedprior to use. Lyophilizing may result in a more stable composition(vaccine) and may possibly lead to higher antibody titers in thepresence of 3D-MPL and in the absence of an aluminium based adjuvant.

In one aspect of the invention is provided a vaccine kit, comprising avial containing an immunogenic composition of the invention, optionallyin lyophilised form, and further comprising a vial containing anadjuvant as described herein. It is envisioned that in this aspect ofthe invention, the adjuvant will be used to reconstitute the lyophilisedimmunogenic composition.

Although the vaccines of the present invention may be administered byany route, administration of the described vaccines into the skin (ID)forms one embodiment of the present invention. Human skin comprises anouter “horny” cuticle, called the stratum corneum, which overlays theepidermis. Underneath this epidermis is a layer called the dermis, whichin turn overlays the subcutaneous tissue. Researchers have shown thatinjection of a vaccine into the skin, and in particular the dermis,stimulates an immune response, which may also be associated with anumber of additional advantages. Intradermal vaccination with thevaccines described herein forms a preferred feature of the presentinvention.

The conventional technique of intradermal injection, the “mantouxprocedure”, comprises steps of cleaning the skin, and then stretchingwith one hand, and with the bevel of a narrow gauge needle (26-31 gauge)facing upwards the needle is inserted at an angle of between 10-15°.Once the bevel of the needle is inserted, the barrel of the needle islowered and further advanced whilst providing a slight pressure toelevate it under the skin. The liquid is then injected very slowlythereby forming a bleb or bump on the skin surface, followed by slowwithdrawal of the needle.

More recently, devices that are specifically designed to administerliquid agents into or across the skin have been described, for examplethe devices described in WO 99/34850 and EP 1092444, also the jetinjection devices described for example in WO 01/13977; U.S. Pat. No.5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat.No. 5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat. No. 5,569,189, U.S.Pat. No. 5,704,911, U.S. Pat. No. 5,383,851, U.S. Pat. No. 5,893,397,U.S. Pat. No. 5,466,220, U.S. Pat. No. 5,339,163, U.S. Pat. No.5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Pat.No. 5,520,639, U.S. Pat. No. 4,596,556, U.S. Pat. No. 4,790,824, U.S.Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO 97/37705 and WO97/13537. Alternative methods of intradermal administration of thevaccine preparations may include conventional syringes and needles, ordevices designed for ballistic delivery of solid vaccines (WO 99/27961),or transdermal patches (WO 97/48440; WO 98/28037); or applied to thesurface of the skin (transdermal or transcutaneous delivery WO 98/20734;WO 98/28037).

When the vaccines of the present invention are to be administered to theskin, or more specifically into the dermis, the vaccine is in a lowliquid volume, particularly a volume of between about 0.05 ml and 0.2ml.

The content of the immunogenic composition in the skin or intradermalvaccines of the present invention may be similar to conventional dosesas found in intramuscular vaccines (see above). However, it is a featureof skin or intradermal vaccines that the formulations may be “low dose”.Accordingly the protein antigens in “low dose” vaccines are suitablypresent in as little as 0.1 to 10 μg, or 0.1 to 5 μg per dose; and thepolysaccharide (suitably conjugated) antigens may be present in therange of 0.01-1 μg, and suitably between 0.01 to 0.5 μg of saccharideper dose.

As used herein, the term “intradermal delivery” means delivery of thevaccine or immunogenic composition to the region of the dermis in theskin. However, the vaccine or immunogenic composition will notnecessarily be located exclusively in the dermis. The dermis is thelayer in the skin located between about 1.0 and about 2.0 mm from thesurface in human skin, but there is a certain amount of variationbetween individuals and in different parts of the body. In general, itcan be expected to reach the dermis by going 1.5 mm below the surface ofthe skin. The dermis is located between the stratum corneum and theepidermis at the surface and the subcutaneous layer below. Depending onthe mode of delivery, the vaccine or immunogenic composition mayultimately be located solely or primarily within the dermis, or it mayultimately be distributed within the epidermis and the dermis.

The present invention further provides an improved vaccine for theprevention or amelioration of otitis media caused by Haemophilusinfluenzae by the addition of Haemophilus influenzae proteins, forexample protein D in conjugated form. One or more Moraxella catarrhalisprotein antigens can also be included in the vaccine or immunogeniccomposition of the invention in a free or conjugated form. Thus, thepresent invention is an improved method to elicit an immune responseagainst otitis media in infants.

Examples of preferred Moraxella catarrhalis protein antigens which canbe included in a combination vaccine or immunogenic composition of theinvention (especially for the prevention of otitis media) are: outermembrane protein 106 (OMP106) [WO 97/41731 (Antex) & WO 96/34960 (PMC)];outer membrane protein 21 (OMP21) or fragments thereof (WO 0018910);lactoferrin binding protein A (LbpA) &/or lactoferrin binding protein B(LbpB) [WO 98/55606 (PMC)]; transferrin binding protein A (TbpA) &/ortransferring binding protein B (TbpB) [WO 97/13785 & WO 97/32980 (PMC)];Moraxella catarrhalis CopB protein [Helminen M E, et al. (1993) Infect.Immun. 61:2003-2010]; ubiquitous surface protein A1 (UspA1) and/orubiquitous surface protein A2 (UspA2) [WO 93/03761 (University ofTexas)]; outer membrane protein CD (OmpCD); HasR (PCT/EP99/03824); PilQ(PCT/EP99/03823); outer membrane protein 85 (OMP85) (PCT/EP00/01468);lipo06 (GB 9917977.2); lipo10 (GB 9918208.1); lipo11 (GB 9918302.2);lipo18 (GB 9918038.2); outer membrane protein P6 (P6) (PCT/EP99/03038);D15 surface antigen (D15) (PCT/EP99/03822); outer membrane protein A1(OmpA1) (PCT/EP99/06781); Hly3 (PCT/EP99/03257); and outer membraneprotein E (OmpE). Examples of non-typeable Haemophilus influenzaeproteins or fragments thereof which can be included in a combinationvaccine (especially for the prevention of otitis media) include: Fimbrinprotein [(U.S. Pat. No. 5,766,608—Ohio State Research Foundation)] andfusions comprising peptides therefrom [eg LB1(f) peptide fusions; U.S.Pat. No. 5,843,464 (OSU) or WO 99/64067]; outer membrane protein 26(OMP26) [WO 97/01638 (Cortecs)]; P6 [EP 281673 (State University of NewYork)]; TbpA and/or TbpB; H. influenzae adhesin (Hia); Haemophilussurface fibrils (Hsf); Haemophilus influenzae Hin47 protein; Haemophilusinfluenzae Hif protein; Haemophilus influenzae Hmw1 protein; Haemophilusinfluenzae Hmw2 protein; Haemophilus influenzae Hmw3 protein;Haemophilus influenzae Hmw4 protein; Haemophilus influenzaeautotransporter adhesin (Hap); D15 (WO 94/12641); P2; and P5 (WO94/26304).

Methods of Treatment and Use

The present invention provides a method for the treatment or preventionof Streptococcus pneumoniae infection in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of an immunogenic composition or the vaccine of the invention.The present invention also provides a method of immunising a human hostagainst Streptococcus pneumoniae infection comprising administering tothe host an immunoprotective dose of the immunogenic composition orvaccine of the invention. The present invention also provides a methodof inducing an immune response to Streptococcus pneumoniae (e.g.Streptococcus pneumoniae serotype 6A) in a subject, the methodcomprising administering a therapeutically effective amount of theimmunogenic composition or vaccine of the invention.

In an embodiment, the present invention is an improved method to elicitan immune response in infants (defined as 0-2 years old in the contextof the present invention) by administering a therapeutically effectiveamount of an immunogenic composition or vaccine of the invention. In oneembodiment, the immune response is protective (i.e. it can prevent orreduce infection caused by S. pneumoniae). In one embodiment, thevaccine is a paediatric vaccine.

In an embodiment, the present invention is an improved method to elicita (protective) immune response in the elderly population (in the contextof the present invention a patient is considered elderly if they are 50years or over in age, typically over 55 years and more generally over 60years) by administering a therapeutically effective amount of theimmunogenic composition or vaccine of the invention.

In one embodiment, the present invention provides a method of protectinga subject against a disease caused by infection with Streptococcuspneumoniae, or a method of preventing infection with Streptococcuspneumoniae, or a method of reducing the severity of or delaying theonset of at least one symptom associated with an infection caused byStreptococcus pneumoniae, the methods comprising administering to asubject an immunogenic amount of an immunogenic composition or vaccineof the invention.

In an embodiment, the present invention provides immunogeniccompositions and vaccines of the invention for use in the prevention ortreatment of a disease caused by S. pneumoniae infection. In anembodiment, the present invention provides the use of an immunogeniccomposition or vaccine of the invention in the manufacture of amedicament for the prevention (or treatment) of a disease caused by S.pneumoniae infection.

The disease caused by Streptococcus pneumoniae infection may be selectedfrom pneumonia, invasive pneumococcal disease (IPD), exacerbations ofchronic obstructive pulmonary disease (COPD), otitis media, meningitis,bacteraemia, pneumonia and/or conjunctivitis. Where the human host is aninfant, the disease may be selected from otitis media, meningitis,bacteraemia, pneumonia and/or conjunctivitis. Where the human host iselderly, the disease may be selected from pneumonia, invasivepneumococcal disease (IPD), and/or exacerbations of chronic obstructivepulmonary disease (COPD).

Embodiments herein relating to “vaccine compositions” of the inventionare also applicable to embodiments relating to “immunogeniccompositions” of the invention, and vice versa.

Embodiments of the invention are further described in the subsequentnumbered paragraphs:

Paragraph 1: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide wherein the average size (M_(w)) of the Streptococcuspneumoniae serotype 6A capsular polysaccharide is between 180-400,210-400, 210-370, 220-360, 230-350, 240-340, 240-320, 240-310 or 250-310kDa.

Paragraph 2: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to paragraph 1 which has been sized by amechanical sizing technique.

Paragraph 3: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to paragraph 1 or paragraph 2 conjugated to acarrier protein (e.g. CRM-197).

Paragraph 4: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to paragraph 3 wherein the Streptococcuspneumoniae serotype 6A capsular polysaccharide is directly conjugated tothe carrier protein.

Paragraph 5: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to any one of paragraphs 3-4 wherein the 6Apolysaccharide is conjugated to the carrier protein or to a linker usingCDAP chemistry.

Paragraph 6: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to any one of paragraphs 3-5 wherein theserotype 6A capsular polysaccharide (PS6A) is conjugated to the carrierprotein or to a linker using CDAP chemistry using a CDAP:PS6A ratiobetween 1:2 to 3:1, 1:1.5 to 2:1, or 1:1.

Paragraph 7: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to any one of paragraphs 3-6 wherein theserotype 6A capsular polysaccharide is conjugated to the carrier proteinor to a linker using CDAP chemistry wherein the reaction was carried outusing a coupling time of between 50-130 minutes, 60-130 minutes, or110-130 minutes.

Paragraph 8: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to any one of paragraphs 3-7 wherein the ratioof carrier protein to serotype 6A capsular polysaccharide is between 5:1and 1:5, 4:1 and 1:1 or 2:1 and 1:1, 1.5:1 and 1:1, 1.4:1 and 1.3:1 (forexample 1.2:1, 1.5:1) (w/w).

Paragraph 9: A sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to paragraphs 1-8 wherein the 6A capsularpolysaccharide is sized by a factor of no more than ×5.

Paragraph 10: A process for preparing a Streptococcus pneumoniaeserotype 6A capsular polysaccharide conjugate comprising conjugating asized Streptococcus pneumoniae serotype 6A capsular polysaccharide to acarrier protein or to a linker using CDAP chemistry using a CDAP:PSratio between 1:2 to 3:1, 1:1.5 to 2:1, or 1:1.

Paragraph 11: A process according to paragraph 10 wherein the reactionwas carried out using a coupling time of between 50-130 minutes, 60-130minutes, or 110-130 minutes.

Paragraph 12: A process for preparing a Streptococcus pneumoniaeserotype 6A capsular polysaccharide conjugate comprising (a) conjugationof a sized Streptococcus pneumoniae serotype 6A capsular polysaccharideto a carrier protein and (b) diafiltration against a solution having aconcentration of NaCl below 150 mM.

Paragraph 13: An immunogenic composition comprising a sizedStreptococcus pneumoniae serotype 6A capsular polysaccharide accordingto paragraphs 1-9 or Streptococcus pneumoniae serotype 6A capsularpolysaccharide obtained by a process of any one of paragraphs 10-12conjugated to a carrier protein.

Paragraph 14: An immunogenic composition according to paragraph 13comprising 10 or more, 11 or more, 12 or more, 13 or more, 14 or more,15 or more, or 16 or more capsular polysaccharides conjugates fromdifferent S. pneumoniae serotypes.

Paragraph 15: An immunogenic composition according to paragraph 13 orparagraph 14 comprising 1 or more, native capsular polysaccharidesconjugates from S. pneumoniae.

Paragraph 16: An immunogenic composition according to paragraph 15comprising native Streptococcus pneumoniae capsular serotype 6Bpolysaccharide.

Paragraph 17: An immunogenic composition according to paragraph 15 or 16comprising native Streptococcus pneumoniae capsular serotype 23Fpolysaccharide.

Paragraph 18: An immunogenic composition according to any one ofparagraphs 13 to 17 which comprises Streptococcus pneumoniae capsularserotype 6B polysaccharide having an average size (M_(w)) of between500-1800, 900-1660, or 1000-1400 kDa.

Paragraph 19: An immunogenic composition according to any one ofparagraphs 13 to 18 which comprises Streptococcus pneumoniae capsularserotype 23F polysaccharide having an average size (M_(w)) of between500-1500, 600-1500, 700-1300, 900-1250, 800-1100, or 900-1000 kDa.

Paragraph 20: An immunogenic composition according to any one ofparagraphs 13 to 19 which comprises Streptococcus pneumoniae capsularpolysaccharide from: (a) serotype 1 having an average size (M_(w)) ofbetween 100-1000, 200-800, 250-600, or 300-400 kDa; (b) serotype 4having an average size (M_(w)) of between 50-500, 60-300, 70-150, or75-125 kDa; (c) serotype 5 having an average size (M_(w)) of between100-1000, 100-700, 100-350, or 150-300 kDa; (d) serotype 7F having anaverage size (M_(w)) of between 50-1000, 100-750, 150-500, or 200-300kDa; (e) serotype 9V having an average size (M_(w)) of between 50-1000,100-750, 150-500, 200-400, or 250-300 kDa; (f) serotype 14 having anaverage size (M_(w)) of between 50-1000, 100-750, 150-500, or 200-250kDa; (g) 18C having an average size (M_(w)) of between 50-1000, 50-750,50-500, 50-190, 50-150 or 80-110 kDa (h) serotype 19F having an averagesize (M_(w)) of between 50-1000, 100-750, 100-500, 100-190 or 120-180kDa; and/or (i) serotype 19A having an average size (M_(w)) of between50-800 kDa, 110-700, 110-300, 120-200, 130-180, 140-160 or 80-130 kDa.

Paragraph 21: An immunogenic composition according to any one ofparagraphs 13 to 20 which further comprises Streptococcus pneumoniaecapsular serotype 22F having an average size (M_(w)) of between 50 and800 kDa, 110 and 700 kDa, 110-300, 120-200, 130-180, 150-170, 100-190,100-150, 95-125 or 100-115 kDa.

Paragraph 22: An immunogenic composition according to any one ofparagraphs 13 to 21 comprising 2, 3, 4, 5 or 6 different carrierproteins.

Paragraph 23: An immunogenic composition according to any one ofparagraphs 13 to 22 wherein one or more or all carrier proteins isselected from the group consisting of: diphtheria toxoid (DT), CRM197,tetanus toxoid (TT), Fragment C of TT, dPly, PhtA, PhtB, PhtD, PhtE,PhtDE OmpC, PorB and Haemophilus influenzae Protein D.

Paragraph 24: An immunogenic composition of any one of paragraphs 13 to23 wherein the Streptococcus pneumoniae capsular serotype 6Bpolysaccharide is conjugated to a different carrier protein (e.g.protein D) than the carrier protein to which Streptococcus pneumoniaeserotype 6A capsular polysaccharide is conjugated.

Paragraph 25: An immunogenic composition of any one of paragraphs 13 to24 comprising serotype 1 saccharide conjugated to protein D, serotype 4saccharide conjugated to protein D, serotype 5 saccharide conjugated toprotein D, serotype 6B saccharide conjugated to protein D, serotype 7Fsaccharide conjugated to protein D, serotype 9V saccharide conjugated toprotein D, serotype 14 saccharide conjugated to protein D and serotype23F saccharide conjugated to protein D.

Paragraph 26: An immunogenic composition according to any one ofparagraphs 13 to 25 comprising serotype 19F conjugated to Diphtheriatoxoid.

Paragraph 27: An immunogenic composition according to any one ofparagraphs 13 to 26 wherein the composition comprises capsularsaccharide 18C conjugated to tetanus toxoid (TT), optionally wherein 18Cis the only saccharide in the composition conjugated to tetanus toxoid(TT), optionally via an ADH linker

Paragraph 28: An immunogenic composition according to any of paragraphs13 to 27 further comprising S. pneumoniae capsular saccharide(s) of oneor more of: serotype 33F, serotype 15 and serotype 12F, conjugated tocarrier protein(s).

Paragraph 29: An immunogenic composition according to any of paragraphs13 to 28 wherein the dose of the 6A saccharide conjugate is between 1and 10 μg, 1 and 5 μg, or 1 and 3 μg of saccharide (e.g. 2 μg).

Paragraph 30: An immunogenic composition according to any one ofparagraphs 13 to 29 which further comprises one or more unconjugated orconjugated S pneumoniae proteins selected from: Poly Histidine Triadfamily (PhtX), Choline Binding Protein family (CbpX), CbpX truncates,LytX family, LytX truncates, CbpX truncate-LytX truncate chimericproteins, detoxified pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130,Sp125 and Sp133.

Paragraph 31: An immunogenic composition according to any one ofparagraphs 13 to 30 which further comprises an adjuvant.

Paragraph 32: An immunogenic composition of paragraph 31 wherein theadjuvant comprises (per 0.5 mL dose) 100-750, 200-500, or 300-400 μg Al(aluminium) as aluminium phosphate.

Paragraph 33: A vaccine comprising the immunogenic composition of anyone of paragraphs 13 to 32 and a pharmaceutically acceptable excipientor carrier.

Paragraph 34: A process for making the vaccine according to paragraph 33which comprises the step of mixing the immunogenic composition of any ofparagraphs 13 to 32 with a pharmaceutically acceptable excipient orcarrier.

Paragraph 35: A method for the treatment or prevention of Streptococcuspneumoniae infection in a subject in need thereof comprisingadministering to said subject a therapeutically effective amount of animmunogenic composition of any of paragraphs 13 to 32 or the vaccine ofparagraph 33.

Paragraph 36: A method of immunising a human host against Streptococcuspneumoniae infection comprising administering to the host animmunoprotective dose of the immunogenic composition of any ofparagraphs 13 to 32 or vaccine of paragraph 34.

Paragraph 37: A method of inducing an immune response to Streptococcuspneumoniae serotype 6A in a subject, the method comprising administeringa therapeutically or prophylactically effective amount of theimmunogenic composition of any of paragraphs 13 to 32 or the vaccine ofparagraph 33.

Paragraph 38: The immunogenic composition of paragraphs 13 to 32 orvaccine of paragraph 33 for use in the treatment or prevention ofdisease caused by Streptococcus pneumoniae infection.

Paragraph 39: A use of the immunogenic composition of paragraphs 12 to32 or vaccine of paragraph 33 in the manufacture of a medicament for thetreatment or prevention of a disease caused by Streptococcus pneumoniaeinfection.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES Example 1—Preparation of Sized Polysaccharide

Sizing

A homogenizer EMULSIFLEX C-50 apparatus was used to reduce the molecularweight and the viscosity of the polysaccharide before the activationstep (microfluidization). The efficiency of the sizing depended on thecircuit pressure and on the total cycle's number. The homogenizing cellof EMULSIFLEX C-50 was replaced by a cell with a fixed geometry(Microfluidics F20Y-75 μm interaction chamber for E01 to E07;Microfluidics F30Y-125 μm interaction chamber for E08). The aim of thesizing was to reduce the molecular weight and the viscosity of thepolysaccharide without a critical decrease of its antigenicity.

The size reduction was followed in-process by viscosimetry (BrookfieldProgrammable DV III+rheometer). When the target in viscosity wasreached, the sized polysaccharide was characterized by HP-SEC-RI(high-performance size-exclusion chromatography, refractive index).

In-process characterizations of the M_(w) were performed by HP-SEC-RI(TSK5000PW_(XL) column+guard column). The elution was realised using a50 mM Na/K₂PO₄, 500 mM NaCl pH 7.6 at a flow rate of 0.6 ml/min. Thedetection was realised by a refractive index detector. Thischaracterization is based on the determination of a relative retentiontime (RRT). The RRT is calculated with regard to calibrated dextranshaving a molecular weight in the linear range of separation of aTSK5000PW_(XL) column. The RRT is defined by the following formula:

${RRT} = \frac{{RT}_{({{sized}\mspace{11mu} {PS}})} - {RT}_{({{HMW}\mspace{11mu} {dext}})}}{{RT}_{({{LMW}\mspace{11mu} {dext}})} - {RT}_{({{HMW}\mspace{11mu} {dext}})}}$

RT_((sized PS))=retention time of sized PS (polysaccharide)

RT_((HMW dext))=retention time of high molecular weight dextran (dextran1730 Kda)

RT_((LMW dext))=retention time of low molecular weight dextran (dextran150 Kda)

The native serotype 6A polysaccharide was dissolved at 15 mg/ml during 4hours in WFI (water for injection) at room temperature. After 4 hours ofdissolution, the pH of the solution was adjusted at 6.5+/−0.5 before itstransfer in a cold room to pursue dissolution overnight. Before thesizing, the solution of native serotype 6A polysaccharide was clarifiedon 5 μm filter.

The serotype 6A polysaccharide was then sized by Emulsiflex with aMicrofluidics F30Y-125 μm homogenizing cell at a pressure of between2900 and 3800. The sizing was stopped when the viscosity of thepolysaccharide reached the targeted value for the viscosity (8.35 or12.4). The number of cycles required depended on the target. Forexample, 32.5 cycles were needed to reach a viscosity of 12.4 cps usinga pressure of 2900 psi. For this sample, in process determination ofrelative retention time by HP-SEC-RI gave a value of 0.28.

The sized 6A polysaccharide was filtered on a Millipak 20 (5 g scale)membrane (cut-off 0.22 μm) at a flow-rate of 10 ml/min.

The polysaccharide content of sized serotype 6A polysaccharide solutionwas accurately determined by colorimetric method (resorcinol) before itsuse in conjugation. Sized polysaccharide was characterized either byHP-SEC-MALLS or estimated by dextrans calibration curve anddetermination of antigenic activity (Table 1).

Antigenicity

Test and reference samples were incubated in microtiter platespreviously coated with monoclonal antibodies raised against S.pneumoniae polysaccharide serotype 6A (PS6A). Rabbit polyclonalanti-PS6A antibodies were then added. Antigen-antibody complex wererevealed using a goat anti-rabbit Ig peroxidase linked. Colourdevelopment was then performed by the system Ortho-phenylenediamine/H₂O₂reacting with peroxidase. Coloration was measured by spectrophotometry(absorbances at 490 and 620 nm). The polysaccharide curve was comparedto the reference curve (one native polysaccharide lot used as referencewhile another one native polysaccharide lot was used as internalcontrol) in order to determine the polysaccharide concentration.

Measurement of Molecular Weight and Polydispersity by MALLS

The molecular weight (M_(w)) was determined by Laser Light Scattering(SEC-MALLS). In a first time, the analyses were performed on aTSK5000PW_(XL) (+guard column) using NaCl 0.2M+0.02% azide solution aselution buffer. Last analyses were performed on a TSKGMPW_(XL) column(+guard column) with a loading of 100 μl of polysaccharide (1 mg/ml)using 50 mM Na/K₂PO₄, 200 mM NaCl pH 7.0 as elution buffer and aflow-rate of 0.75 ml/min.

The detection was realised with a laser spectrophotometer and aninterferometric refractometer (Wyatt Otilab DSP equipped with a P100cell and a red filter at 498 nm).

The average molecular weight in number (M_(n)) was also obtained byMALLS. The polydispersity of the sized polysaccharide was obtained asthe M_(w)/M_(n) ratio.

A theoretical do/dc of 0.14 was first used. When determined, theexperimental value of 0.151 was used.

TABLE 1 Sized PS6A characterizations M_(W) (kDa) ² (estimation MALLSversus dextran M_(W) (kDa) PS6A PS content RRT Viscosity calibrationR_(w) (nm) Antigenicity by Lot (μg/ml) 150-1730 ¹ (cp) curve)Polydispersity ELISA (%) E01 13650 0.45 8.35 575 ND 124 E02  8570 0.506.03 514 ND ND E03 14210 0.24 15.6 968 ND ND E04 12000 0.21 17.3 1018120 ³ E05 — — — — — — E06 13910 0.24 12.1 916 T13M/−70° C. 130 ³ 25625.6 1.124 E07 14700 0.24 12.2 929 307.4 120 ³ 28.1 1.112 E08 13590 0.2012.3 1004 T12M/−70° C. 144 ³ 277 27.7 1.094 ¹ RRT: relative retentiontime (HP-SEC-RI) ² HP-SEC-RI ³ relative value versus correspondingnative polysaccharide. Remark: No MALLS values available at T0 for lotsE06 and E08. Value at T13M (time = 13 minutes) or T12M (time = 12minutes) could be considered as representative insofar as relativeretention time and viscosity did not change between T0 (time = 0minutes) and T13M or T12M for both samples. ND means ″not determined″.

Example 2: Production of 6A Conjugates with Different Carriers

a) Native S. pneumoniae Serotype 6A Polysaccharide Vs. Sized S.pneumoniae Serotype 6A Polysaccharide

The activation and coupling conditions used to produce different 6Aconjugates are given in Table 2.

TABLE 2 Specific activation/coupling/quenching conditions of S.pneumoniae serotype 6A polysaccharide - Protein D/PhtD conjugates PD(lot PhtD (lot PhtD (lot dPly (lot PD003) PhtD004) PhtD008) dPly010)(PS6A-PD (PS6A-PhtD (PS6A-PhtD (PS6A-dPly Carrier conjugate) conjugate)conjugate) conjugate) PS6A conc. 5.5 5.5 10.0 10 (mg/ml) Native PSNative PS Sized PS Sized PS PS6A NaCl 2M NaCl 2M NaCl 2M NaCl 2Mdissolution Carrier protein 5.0  10.0  20.0  10.0  concentration (mg/ml)Initial Carrier 1/1 3/1 3/1 3/1 protein/PS6A Ratio (w/w) CDAP/PS6A 0.751.5 1.5 1.5 ratio (mg/mg PS) Coupling time 60 min 45 min 150 min 180 minpH_(a)/pH_(c)/pH_(q) 9.5/9.5/9.0 9.5/9.5/9.0 9.5/9.5/9.0 9.5/9.5/9.0

The final protein/PS6A ratio (w/w) for the resulting conjugates were:

PS6A-PD003: 0.6/1 (Native PS6A, M_(w) by MALLS 1106 kDa)

PS6A-PhtD004: 1.4/1 (Native PS6A, M_(w) by MALLS 1106 kDa)

PS6A-PhtD008: 2.7/1 (Sized PS6A, lot E01)

PS6A-dPly010: 1.65/1 (Sized PS6A, lot E01)

b) Sized Polysaccharide Conjugated to Different Carriers

The activation and coupling conditions used to produce different PS6Aconjugates are given in Table 3.

TABLE 3 Specific activation/coupling/quenching conditions ofPS6A-Protein D/CRM197/PhtD conjugates PD (lot PDLS001 and CRM197 (lotCRM025) lot PDLS002) (PS6A-CRM197 Carrier (PS6A-PD conjugate) conjugate)PS6A conc. 10.0 10.0 (mg/ml) PS6A dissolution NaCl 2M NaCl 2M Carrierprotein 10.0 10.0 concentration (mg/ml) Initial Carrier protein/ 1.5/11.5/1 PS6A Ratio (w/w) CDAP conc.  1.5 1   (mg/mg PS) Coupling time 120min 120 min pH_(a)/pH_(c)/pH_(q) 9.5/9.5/9.0 9.5/9.5/9.0

The final protein/PS6A ratio (w/w) for the resulting conjugates were:

PS6A-PD/LS001 and PS6A-PD/LS002: 1.6/1 (Sized PS6A, lot E06)

PS6A-CRM197/025: 1.3/1 (Sized PS6A, lot E03)

PS6A_(AH)-PhtD conjugate (lot 6A-PhtD106)

In a second conjugation method PS6A was linked to the carrier proteinPhtD via a linker—Adipic acid dihydrazide (ADH); this conjugate isdesignated PS6A_(AH)-PhtD.

PS6A Derivatization

Activation and coupling were performed at 25° C. under continuousstirring in a temperature-controlled waterbath. Microfluidized PS6A wasdiluted to obtain a final polysaccharide concentration of 10 mg/ml inwater for injection (WFI) and the solution was adjusted at pH 6.0±0.2with 0.1N HCl. CDAP solution (100 mg/ml freshly prepared inacetonitrile/WFI, 50/50) was added to reach the appropriate CDAP/PSratio (1/1, w/w).

The pH was raised up to the activation pH 9.00±0.05 by the addition of0.2M NaOH. After 3 minutes, ADH was added to reach the appropriateADH/PS ratio (8.9/1 w/w); the pH was regulated to coupling pH 9.0. Thesolution was left for 1 hour under pH regulation. The PSAH derivativewas then dialysed against 0.2M NaCl.

Coupling

PhtD at 7.5 mg/ml in 0.2M NaCl was added to the PS6A_(AH) derivative(PS6A with the ADH linker) in order to reach a PhtD/PS6A_(AH) ratio of3/1 (w/w). The pH was adjusted to 5.0±0.05 with HCl. The EDAC solution(50 mg/ml in 0.1M Tris-HCl pH 7.5) was added manually in 10 min (20μl/min) to reach 0.5 mg EDAC/mg PS6A_(AH). The resulting solution wasincubated for 45 min at room temperature under stirring and pHregulation. The solution was neutralized by addition of 1 ml of 1MTris-HCl pH 7.5 and let 30 min at room temperature.

Prior to the elution on Sephacryl S400HR, the conjugate was clarifiedusing a 5 μm Minisart filter. The resulting conjugate PS6A_(AH)-PhtD106had a final PhtD/PS6A ratio of 2.84 (w/w).

A target in relative retention time was chosen (RRT₁₅₀₋₁₇₃₀ around0.50). Several conjugates (6A-PhtD004 (native polysaccharide) and6A-PhtD008 (sized polysaccharide) (as described above) were producedwith sized polysaccharide and were compared to conjugates produced withnative PS6A.

Example 3: Preclinical Evaluation of Anti-PS6A Responses

Groups of 40 mice were immunized IM at days 0, 14 and 28 with 14 valent(14V) formulations containing PS6A conjugates (at 1/10 of human dose)using AlPO₄ as adjuvant. Anti-PS6A ELISA IgG titers andOpsonophagocytosis (OP) titers were measured in individual seracollected at day 42.

The 14V-formulation contained the following conjugates:

1-PD, 3-PD, 4-PD, 5-PD, 6B-PD, 7F-PD, 9V-PD, 14-PD, 18C-TT_(AH),19A-dPly, 19F-DT, 22F-PhtD, 23F-PD. The serotype 6A was conjugated withPD, PhtD or dPly as carrier.

Results are summarized in FIG. 1 ELISA anti-PS6A response.

Conclusions:

The highest anti-PS6A ELISA IgG titers were obtained with the PS6A-PDconjugate produced with a native polysaccharide (1106 kDa). Theevaluation showed a trend to a lower immunogenicity for conjugates withsized polysaccharide. Using PhtD as carrier, a lower anti-PS ELISA IgGtiter was observed with the conjugate produced with a sizedpolysaccharide (14V (6A-PhtD/008 using lot E01). Subsequently, asdescribed in the following examples, a change in sizing target wasinvestigated to produce a PS6A with robustness of process andimmunogenicity of resulting conjugates. The following experiments werecarried out to evaluate sized polysaccharide having a higher molecularweight than PS6A from lot E01.

Example 4: Evaluation of Sized PS6A-CRM197 Conjugates

For each conjugate, conjugation parameters are given in Table 4 (PS6Aand carrier concentrations, initial carrier/PS6A ratio (w/w), couplingtime and CDAP/PS6A ratio (w/w)

Preparation of the Solutions

CDAP Solution

Just before activation, a cyanodiaminopyridinium tetrafluoroborate(CDAP) solution was prepared at 100 mg/ml in acetonitrile/water forinjection (50/50 (v/v))

Activation

Native or sized PS6A was diluted at a defined concentration and the pHof the solution was set to 6.0±0.2. At time 0, the CDAP solution wasadded manually in order to obtain a defined CDAP/PS6A ratio (w/w).

After 1.5 minutes the pH was raised up to activation pH value byaddition of NaOH. At time 4.5 minutes, the protein solution (at adefined concentration and buffer) was added in order to obtain a fixedprotein/PS6A ratio (w/w). The pH of the solution was regulated atcoupling pH value during a defined timing (coupling time—See Table 4).At time T=coupling time+4 min 30, a solution of Glycine 2M pH 9.0 wasadded to quench the reaction. After 30 minutes of quenching, theconjugate was directly injected on purification column or let overnightunder continuous stirring at +2 to +8° C. before purification.

Purification

Before purification, the conjugate solution was filtered through a 5 μmor 10 μm membrane in order to remove aggregates and particles. Conjugatewas then purified on Sephacryl S400HR column (bed height: 100 cm+/−10cm) using NaCl 150 mM as eluent. The conjugate was then sterile filteredon 0.22 μm PVDF (polyvinylidene difluoride) membrane. The sterilisedbulk (Conjugated Bulk) was stored at +2-8° C. until formulation.

Characterization

The final Protein/Polysaccharide ratio (w/w) on the sterilized conjugatewas determined by the ratio of the Lowry/resorcinol concentrations.Antigenicity and free PS (polysaccharide) content were determined usingmethods described here below. Data are shown in Table 5.

Antigenicity Test

Native polysaccharide was assigned an antigenicity index value of 100%.Insofar as correlation between antigenicity index and immunogenicity hadnot been established, the determination of sized polysaccharideantigenicity was performed as indicative value. The objective was tokeep this value as high as possible. This value was determined by ELISA.

The antigenicity of polysaccharide was determined in a sandwich-typeELISA (see Example 1 for measurement of antigenicity).

Free PS Content by ELISA

After reaction of the conjugate with anti-carrier serum, the complex wasprecipitated using saturated ammonium sulphate (SAS). Aftercentrifugation, the free PS06A content was performed by ELISA(anti-PS/anti-PS, see part 2.8.1.2) on the supernatant. The percentageof free PS was calculated proportionally to the total PS contentmeasured by resorcinol method.

The absence of conjugate in the supernatant was also controlled by aα-carrier/α-PS06A ELISA.

TABLE 4 Process conditions for PS6A-CRM197 conjugates at 50 mg scaleCarrier PS Dissolution Dissolution Conjugate PS6A (M NaCl) [mg/ml]buffer [mg/ml] PS6A- native 2 5 K/K₂PO₄ 10 mM 10 CRM197/015 pH7 2NaCl0.2M PS6A- native 2 5 K/K₂PO₄ 10 mM 10 CRM197/016 pH7 2NaCl 0.2M PS6A-E01 2 10 K/K₂PO₄ 10 mM 10 CRM197/017 pH7 2NaCl 0.2M PS6A- E01 2 10K/K₂PO₄ 10 mM 10 CRM197/018 pH7 2NaCl 0.2M PS6A- E01 2 10 K/K₂PO₄ 10 mM10 CRM197/019 pH7 2NaCl 0.2M PS6A- E01 2 10 K/K₂PO₄ 10 mM 10 CRM197/020pH7 2NaCl 0.2M PS6A- E01 2 10 K/K₂PO₄ 10 mM 10 CRM197/021 pH7 2NaCl 0.2MPS6A- E03 2 10 K/K₂PO₄ 10 mM 10 CRM197/022 pH7 2NaCl 0.2M PS6A- E03 2 10K/K₂PO₄ 10 mM 10 CRM197/023 pH7 2NaCl 0.2M PS6A- E03 2 10 K/K₂PO₄ 10 mM10 CRM197/024 pH7 2NaCl 0.2M Initial CDAP Coupling pHa/ Ratio (mg/mgTime pHc/ Conjugate (w/w) PS) (min) PHq Remark PS6A- 1.5/1 1.5/1 609.5/9.5/9.0 50 mg CRM197/015 PS6A- 1.5/1 0.75/1  60 9.5/9.5/9 50 mgCRM197/016 PS6A- 1.5/1 0.75/1  120 9.5/9.5/9 50 mg CRM197/017 PS6A-1.5/1 1.5/1 120 9.5/9.5/9 50 mg CRM197/018 PS6A- 1.5/1   1/1 1209.5/9.5/9 50 mg CRM197/019 PS6A-   2/1 0.75/1  120 9.5/9.5/9 50 mgCRM197/020 PS6A- 1.5/1 1.5/1 120 9.5/9.5/9 200 mg  CRM197/021 PS6A-1.5/1 1.5/1 120 9.5/9.5/9 50 mg CRM197/022 PS6A- 1.5/1 1.5/1 609.5/9.5/9 50 mg CRM197/023 PS6A- 1.5/1   1/1 120 9.5/9.5/9 50 mgCRM197/024 Note: Designation such as “PS6ACRM197/024” indicatesPS6ACRM197 from lot024.

TABLE 5 Characterization of PS6A-CRM197 conjugates at 50 mg-PS scaleFree Final Ratio polysaccharide αPS/αPS αprot/αPS Carrier/PS ELISA (%)(%) (%) Yield Conjugate (w/w) 4° C. 4° C. 4° C. (%) Remark PS6A- 0.91/158.8 Filtration issue CRM197/015 PS6A- 0.65/1 45.1 CRM197/016 PS6A-1.23/1 28.2 Tightened CRM197/017 pool/bad separation PS6A- 1.33/1 48.3CRM197/018 (NF) PS6A- 1.10/1 50.2 CRM197/019 PS6A- 1.41/1 50.6CRM197/020 PS6A- 1.42/1 0.8 35 125 45.4 CRM197/021 PS6A- 1.11/1 1.1 2367 69.8 Filtration issue CRM197/022 PS6A- 1.24/1 0.8 27 98 61.4CRM197/023 PS6A- 1.19/1 1.3 31 95 65.3 CRM197/024

The first set of conjugates (PS6A-CRM197/015, PS6A-CRM197/016) wereproduced with native polysaccharide (M_(W) by MALLS 990 kDa) and showedlimitations in term of reachable final CRM/PS ratio and filterability ofresulting conjugates (Table 5).

A second set of conjugates (PS6A-CRM197/017, PS6A-CRM197/018,PS6A-CRM197/019, PS6A-CRM197/020, PS6A-CRM197/021) were produced usingsized polysaccharide (lot E01). By increasing CDAP/PS ratio and/orinitial CRM/PS ratio, the desired final CRM/PS ratio and polysaccharideyield could be reached. The best conditions (PS6A-CRM197/021) werereproduced with a new sized polysaccharide lots but having a highermolecular weight (lot E03) than the previous one. However a filtrationissue was observed. It was found that by reducing coupling time orCDAP/PS6A ratio, the filtration issue was solved.

A third set of conjugates (PS6A-CRM197/022, PS6A-CRM197/023,PS6A-CRM197/024) were produced using sized serotype 6A polysaccharide(lot E03). The best conditions (PS6A-CRM197/024) were the following: apolysaccharide concentration of 10 mg/ml in 2M NaCl, a CDAP/PS ratio of1/1 (w/w), a CRM concentration of 10 mg/ml in 10 mM K/K₂ PO₄ pH 7.2,0.2M NaCl, an initial CRM/PS ratio of 1.5/1 (w/w), a pH for activationand coupling of 9.5 and a coupling time of 120 min. Resulting conjugateshad a final CRM/PS ratio around 1.2/1 (w/w) for a global yield around65%.

Example 5: Production of Sized 6A-CRM197 Conjugates at 200 mg Scale

A scale up at 200 mg-PS scale was performed (Table 6 and Table 7).

TABLE 6 Process conditions for PS6A-CRM197 conjugates at 200 mg-PS scaleEstimated molecular PS Carrier weight Dissolution Dissolution [mg/Conjugate Lot (kDa)* (M NaCl) [mg/ml] Lot buffer ml] PS6A- E03 968 2 10CRM197-012 K/K₂PO₄ 10 mM 10 CRM197/025 pH 7 2 NaCl 0.2 M PS6A- E03 968 210 CRM197-012 K/K₂PO₄ 10 mM 10 CRM197/026 pH 7 2 NaCl 0.2 M PS6A- E03968 2 10 CRM197-009 K/K₂PO₄ 10 mM 10 CRM197/027 pH 7 2 NaCl 0.2 M PS6A-E07 929 2 10 CRM197-014 K/K₂PO₄ 10 mM 10 CRM197/028 pH 7 2 NaCl 2 MPS6A- E07 929 2 10 CRM197-014 K/K₂PO₄ 10 mM 10 CRM197/029 pH 7 2 NaCl0.2 M PS6A- E08 1004 2 10 CRM197-014 K/K₂PO₄ 10 mM 10 CRM197/030 pH 7 2NaCl 2 M *Estimation realized using a dextran calibration curve (seeTable 1 for MALLS data) Initial Ratio CouplingTime Conjugate (w/w) CDAP(mg/mg PS) (min) pHa/pHc/pHq PS6A-CRM197/025 1.5/1 1/1 120 9.5/9.5/9PS6A-CRM197/026 1.5/1 1/1 120 9.5/9.5/9 PS6A-CRM197/027 1.5/1 1/1 1209.5/9.5/9 PS6A-CRM197/028 1.5/1 1/1 120 9.5/9.5/9 PS6A-CRM197/029 1.5/11/1 120 9.5/9.5/9 PS6A-CRM197/030 1.5/1 1/1 120 9.5/9.5/9

TABLE 7 Characterization of PS6A-CRM conjugates at 200 mg-PS scale FreePS F. Ratio ELISA αPS/αPS αprot/αPS Carrier/PS (%) (%) (%) Conjugate(w/w) 4° C. 4° C. 4° C. Yield (%) PS6A- 1.32/1 0.2 56 132 56.6CRM197/025 PS6A- 1.20/1 0.1 50 125 56.2 CRM197/026 PS6A- 1.25/1 0.2 49113 57.6 CRM197/027 PS6A- 1.18/1 44 89 60.2 CRM197/028 PS6A- 1.22/1 36101 67.8 CRM197/029 PS6A- 1.36/1 28 91 56.4 CRM197/030 Remark E6A-417PS6A- CRM197/ 025

Data from 200 mg-PS scale lots was consistent with data obtained atsmall scale. Resulting conjugates had final CRM/PS ratio between 1.25 to1.32/1, for a global polysaccharide yield of 56%. In term of stability,no issue appeared in HP-SEC analyses or free polysaccharide content byELISA (data not available with chemical method).

Based on these results, further lots (see Table 5 below) were producedin the following conditions: a polysaccharide concentration of 10 mg/mlin 2M NaCl, a CDAP/PS ratio of 1/1 (w/w), a CRM concentration of 10mg/ml in 10 mM K/K₂PO₄ pH 7.2, 2M NaCl, an initial CRM/PS ratio of 1.5/1(w/w), a pH for activation and coupling of 9.5 and a coupling time of120 min.

Example 6: Immunogenicity of PS6A Conjugates in Mice

Different sized PS6A conjugates were evaluated in Balb/c mice (asmonovalent formulation): 6A-CRM197 (PS6A-CRM025), 6A-PD(PS6A-PDLS001(E06)), 6A-PD (PS6A-PDLS002(E06)), 6A-PhtD (PS6A-PhtD008)and 6A_(AH)-PhtD (PS6A-PhtD106 (E04)), PS6A-PhtD (PS6A-PhtD100(E06))conjugates produced with a sized PS.

34 Balb/c Mice were immunized three times (days 0-14-28) with 0.3 μg ofdifferent PS6A conjugates adsorbed onto AlPO₄. Anti-PS6A ELISA IgGtiters levels and Opsonophagocytosis (OP) titers were measured inindividual sera collected at day 42. Results are shown in FIGS. 2A and2B. A significantly higher antibody response was induced by PS6A-AH-PhtD(CDAP/EDAC) and PS6A-CRM 025 (CDAP) in ELISA and OPA.

Example 7: Characterisation of Sized PS6A

CRM197 Concentration

Thawing of CRM197

Purified bulk is stored at −20° C. at a concentration of 1.917 mg/ml in10 mM K/K₂PO₄ pH 7.2. 7.5 g of purified bulk were thawed overnight at+2/+8° C.

Concentration by UF

The ultrafiltration was realized at room temperature on a centramatedevice.

The ultrafiltration membrane was an OMEGA medium screen membrane of 0.09m² with a 10 kDa cut off (2 membranes). The circulation flow-rate was1200 ml/min and the transmembrane pressure applied during the run wasbetween 7-10 psi.

The CRM197 bulk was 7.5-fold concentrated in order to reach aconcentration of around 15 mg/ml (target for conjugation >10 mg/ml).

Filtration on 0.22 μm

After the ultrafiltration, the concentrated bulk was sterile filtered on0.22 μm Millipack 20 filter (PVDF) at 20 ml/min. It was then stored at−20° C. until its use in coupling.

The sized PS6A was characterized by the following test: MALLS andantigen content by ELISA. Stability was evaluated following 2 months at−70° C. (T=0 compared to T=2 months). No stability issue observed whenthe sized PS6A is stored at −70° C. Results of sized PS6Acharacterization are summarized in (Table 8).

TABLE 8 Characterization data and stability of sized PS6A T = 2M at T =0 −70° C. Antigen content by resorcinol (IP value) 15487 μg/ml / Meanmolecular weight in weight by 302.1 kDa 288.8 kDa MALLS Root mean squareradius in weight by 27.7 nm 27.3 nm MALLS Polydispersity ratio 1.1021.136 (M_(w)/Mn) by MALLS Antigen content by ELISA 22250 μg/ml 22911μg/ml Ratio ELISA/resorcinol (%) 144% 148%

Two conjugates (D06ADJA001, D06ADJA002) were produced at 2 g-PS scaleusing lot of sized PS6A and lot as carrier. The conditions ofconjugation are described hereafter.

The reaction was performed at 25+/−1° C. in a 1 L-bioreactor.

2 g of sized PS6A was diluted at 10 mg/ml in 2M NaCl and the pH of thesolution was adjusted at 6.0+/−0.2 with 0.05N HCl solution.

At T=0, 2 g of CDAP in solution (solution at 100 mg/ml in CH₃CN/H₂O50/50) was manually added to the solution (CDAP/PS ratio=1.0/1 w/w).

At T=1 min 30, pH was increased up to 9.5+/−0.05 by addition of 0.5NNaOH. It took approximately 90 sec to reach the target pH.

At T=4 min30, 3 g of CRM197 (solution at 10 mg/ml in 10 mM KH₂PO₄/K₂HPO₄pH 7.2, 2M NaCl) was added at the solution of activated PS6A in 1 minutein order to reach a CRM197/PS6A ratio of 1.5/1 w/w. The pH was regulatedat 9.5+/−0.05 during 120 minutes.

At T=124 min 30, 100 ml of a solution of 2M glycine pH 9.0 was added toquench the conjugate (Ratio Gly/PS=7.5/1 w/w). After 30 minutes ofquenching, the pH of the mixture was adjusted to 6.5+/−0.2 using 5N HCl.When pH was stabilized, the conjugate was let overnight under continuousstirring at +2/+8° C. before clarification and purification.

Purification

Prior to the elution on Sephacryl S400HR, the conjugate was clarifiedusing a 10 μm Kleenpack HDCII filter at 50 ml/min

The conjugate was then injected on a Sephacryl S400HR. Elution was donewith 0.15M NaCl solution and the collection pool was based on a Kdvalue. Kd is the distribution coefficient (Kd=(V_(e)−V₀)/(V_(t)−V)V_(e)=elution volume, V₀=void volume, V_(t)=total volume of the column.

The following criteria were used for the pool collection: fromOD_(280 nm)=0.05 AU to a Kd value of 0.28.

Sterilizing Filtration

Before filtration, the bulk was brought back to room temperature.

Lots were filtered on Opticap 4″ sterilizing membrane (1900 cm²′ PVDF)at a flow-rate of 40 ml/min. The filter was rinsed by 0.15M NaCl bufferbefore filtration. After filtration, 200 ml of 0.15M NaCl was passedthrough filter to limit the loss of material. No issue appeared duringfiltration.

Preclinical Evaluation

Groups of 48 female Balb/c mice (4-weeks-old) were immunized IM at days0, 14 and 28 with adsorbed conjugates formulations (AIPO₄) containing0.1 μg of either D06ADJA001, D06ADJA002, PS6A-CRMLS001 (produced withsized PS6A, lot E07), PS6A-CRMLS002 (produced with sized PS6A, lot E07)or PS6A-PDLS001 (as a benchmark).

Anti-PS6A IgG levels (FIG. 3A) and opsono-phagocytosis titers (FIG. 3B)were measured in individual sera collected at day 42. No significantdifference was observed in antibody responses induced by the PS6A-CRMproduced according to Example 7 compared to the PS6A-CRM producedaccording to Example 5 in ELISA and in OPA.

Example 8: Preparation of 6A Conjugate

Sized 6A conjugate (lot E06AADJA059) was produced at 15 g PS scale in a15 L reactor using the conjugation parameters of the sized 6A producedat 200 mg PS scale (see Example 5). Conjugate was then purified onSephacryl S400HR column (BPG450 column (GE Healthcare)) using NaCl 150mM as eluent. The conjugate was then sterile filtered on 0.22 μm PDVFmembrane and stored at 2-8° C. The conjugate was concentrated (5×) anddiafiltrated (10 diafiltration volume) against WFI (water for injection)using a 10 kDa MWCO OMEGA PES membrane (T-series, PALL). The retentatewas then filtered on 0.22 μm membrane.

SEQ ID NO 1: MetLysLeuLysThrLeuAlaLeuSerLeuLeuAlaAlaGlyValLeuAlaGlyCysSerSerHisSerSerAsnMetAlaAsnThrGlnMetLysSerAspLysIleIleIleAlaHisArgGlyAlaSerGlyTyrLeuProGluHisThrLeuGluSerLysAlaLeuAlaPheAlaGlnGlnAlaAspTyrLeuGluGlnAspLeuAlaMetThrLysAspGlyArgLeuValValIleHisAspHisPheLeuAspGlyLeuThrAspValAlaLysLysPheProHisArgHisArgLysAspGlyArgTyrTyrValIleAspPheThrLeuLysGluIleGlnSerLeuGluMetThrGluAsnPheGluThrLysAspGlyLysGlnAlaGlnValTyrProAsnArgPheProLeuTrpLysSerHisPheArgIleHisThrPheGluAspGluIleGluPheIleGlnGlyLeuGluLysSerThrGlyLysLysValGlyIleTyrProGluIleLysAlaProTrpPheHisHisGlnAsnGlyLysAspIleAlaAlaGluThrLeuLysValLeuLysLysTyrGlyTyrAspLysLysThrAspMetValTyrLeuGlnThrPheAspPheAsnGluLeuLysArgIleLysThrGluLeuLeuProGlnMetGlyMetAspLeuLysLeuVaIGlnLeuIleAlaTyrThrAspTrpLysGluThrGlnGluLysAspProLysGlyTyrTrpValAsnTyrAsnTyrAspTrpMetPheLysProGlyAlaMetAlaGluValValLysTyrAlaAspGlyValGlyProGlyTrpTyrMetLeuValAsnLysGluGluSerLysProAspAsnIleValTyrThrProLeuValLysGluLeuAlaGlnTyrAsnValGluValHisProTyrThrValArgLysAspAlaLeuProGluPhePheThrAspValAsnGlnMetTyrAspAlaLeuLeuAsnLysSerGlyAlaThrGlyValPheThrAspPheProAspThrGlyValGluPheLeuLysGlyIleLys SEQ ID NO. 2:MetAspProSerSerHisSerSerAsnMetAlaAsnThrGlnMetLysSerAspLysIleIleIleAlaHisArgGlyAlaSerGlyTyrLeuProGluHisThrLeuGluSerLysAlaLeuAlaPheAlaGlnGlnAlaAspTyrLeuGluGlnAspLeuAlaMetThrLysAspGlyArgLeuValValIleHisAspHisPheLeuAspGlyLeuThrAspValAlaLysLysPheProHisArgHisArgLysAspGlyArgTyrTyrValIleAspPheThrLeuLysGluIleGlnSerLeuGluMetThrGluAsnPheGluThrLysAspGlyLysGlnAlaGlnValTyrProAsnArgPheProLeuTrpLysSerHisPheArgIleHisThrPheGluAspGluIleGluPheIleGlnGlyLeuGluLysSerThrGlyLysLysValGlyIleTyrProGluIleLysAlaProTrpPheHisHisGlnAsnGlyLysAspIleAlaAlaGluThrLeuLysValLeuLysLysTyrGlyTyrAspLysLysThrAspMetValTyrLeuGlnThrPheAspPheAsnGluLeuLysArgIleLysThrGluLeuLeuProGlnMetGlyMetAspLeuLysLeuVaIGlnLeuIleAlaTyrThrAspTrpLysGluThrGlnGluLysAspProLysGlyTyrTrpValAsnTyrAsnTyrAspTrpMetPheLysProGlyAlaMetAlaGluValValLysTyrAlaAspGlyValGlyProGlyTrpTyrMetLeuValAsnLysGluGluSerLysProAspAsnIleValTyrThrProLeuValLysGluLeuAlaGlnTyrAsnValGluValHisProTyrThrValArgLysAspAlaLeuProGluPhePheThrAspValAsnGlnMetTyrAspAlaLeuLeuAsnLysSerGlyAlaThrGlyValPheThrAspPheProAspThrGlyValGluPheLeuLysGlyIleLys SEQ ID NO. 3:SerSerHisSerSerAsnMetAlaAsnThr

What is claimed is:
 1. A sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide having an average size (M_(w)) between 180-400,210-400, 210-370, 220-360, 230-350, 240-340, 240-320, 240-310 or 250-310kDa.
 2. (canceled)
 3. A sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide according to claim 1 conjugated to a carrierprotein wherein the Streptococcus pneumoniae serotype 6A capsularpolysaccharide is conjugated to a linker or directly conjugated to thecarrier protein and wherein the carrier protein is selected from thegroup consisting of: diphtheria toxoid (DT), CRM197, tetanus toxoid(TT), Fragment C of TT, dPly, PhtA, PhtB, PhtD, PhtE, PhtDE OmpC, PorBand Haemophilus influenzae Protein D.
 4. (canceled)
 5. A sizedStreptococcus pneumoniae serotype 6A capsular polysaccharide accordingto claim 3 wherein the 6A polysaccharide is conjugated to the carrierprotein or to the linker using CDAP chemistry or conjugated to thecarrier protein or to the linker using reductive amination. 6.(canceled)
 7. (canceled)
 8. A sized Streptococcus pneumoniae serotype 6Acapsular polysaccharide according to claim 3 wherein the ratio ofcarrier protein to serotype 6A capsular polysaccharide is between 5:1and 1:5, 4:1 and 1:1, 2:1 and 1:1, 1.5:1 and 1:1, or 1.4:1 and 1.3:1(w/w), and wherein the dose of the 6A capsular polysaccharide conjugateis between 1 and 10 μg, 1 and 5 μg, or 1 and 3 μg of polysaccharide. 9.(canceled)
 10. A process for preparing a Streptococcus pneumoniaeserotype 6A capsular polysaccharide conjugate comprising conjugating asized Streptococcus pneumoniae serotype 6A capsular polysaccharide to acarrier protein or to a linker using CDAP chemistry using a CDAP:PSratio between 1:2 to 3:1, 1:1.5 to 2:1, or a ratio of 1:1 and whereinthe reaction is carried out using a coupling time of between 50-130minutes, 60-130 minutes or 110-130 minutes.
 11. (canceled)
 12. A processfor preparing a Streptococcus pneumoniae serotype 6A capsularpolysaccharide conjugate comprising (a) conjugating a sizedStreptococcus pneumoniae serotype 6A capsular polysaccharide to acarrier protein and (b) diafiltering against a solution having aconcentration of NaCl below 150 mM.
 13. An immunogenic compositioncomprising a sized Streptococcus pneumoniae serotype 6A capsularpolysaccharide according to claim
 3. 14. An immunogenic compositionaccording to claim 13 comprising 10 or more capsular polysaccharidesconjugated to a carrier protein, wherein the 10 or more capsularpolysaccharides conjugated to a carrier protein are from different S.pneumoniae serotypes.
 15. An immunogenic composition according to claim13 comprising 1 or more native serotype capsular polysaccharides from S.pneumoniae conjugated to a carrier protein.
 16. An immunogeniccomposition according to claim 15 comprising native Streptococcuspneumoniae capsular serotype 6B polysaccharide.
 17. (canceled)
 18. Animmunogenic composition according to 13 which comprises Streptococcuspneumoniae capsular serotype 6B polysaccharide having an average size(M_(w)) of between 500-1800, 900-1660, or 1000-1400 kDa.
 19. (canceled)20. An immunogenic composition according to claim 13, further comprisingStreptococcus pneumoniae capsular polysaccharide from: (a) serotype 1having an average size (M_(w)) of between 100-1000, 200-800, 250-600, or300-400 kDa; (b)_serotype 4 having an average size (M_(w)) of between50-500, 60-300, 70-150, or 75-125 kDa; (c) serotype 5 having an averagesize (M_(w)) of between 100-1000, 100-700, 100-350, or 150-300 kDa; (d)serotype 6B polysaccharide having an average size (M_(w)) of between500-1800, 900-1660, or 1000-1400 kDa; serotype 7F having an average size(M_(w)) of between 50-1000, 100-750, 150-500, or 200-300 kDa; (f)serotype 9V having an average size (M_(w)) of between 50-1000, 100-750,150-500, 200-400, or 250-300 kDa; (g) serotype 14 having an average size(M_(w)) of between 50-1000, 100-750, 150-500, or 200-250 kDa; (h) 18Chaving an average size (M_(w)) of between 50-1000, 50-750, 50-500,50-190, 50-150 or 80-110 kDa; (i) serotype 19F having an average size(M_(w)) of between 50-1000, 100-750, 100-500, 100-190 or 120-180 kDa;(j) serotype 19A having an average size (M_(w)) of between 50-800 kDa,110-700, 110-300, 120-200, 130-180, 140-160 or 80-130 kDa; and/or (k)serotype 23F polysaccharide having an average size (M_(w)) of between500-1500, 600-1500, 700-1300, 900-1250, 800-1100, or 900-1000 kDa. 21.An immunogenic composition according to claim 13 which further comprisesStreptococcus pneumoniae capsular serotype 22F having an average size(M_(w)) of between 50 and 800 kDa, 110 and 700 kDa, 110-300, 120-200,130-180, 150-170, 100-190, 100-150, 95-125 or 100-115 kDa.
 22. Animmunogenic composition according to claim 13 comprising 2, 3, 4, 5 or 6different carrier proteins.
 23. (canceled)
 24. An immunogeniccomposition of claim 20 wherein the Streptococcus pneumoniae capsularserotype 6B polysaccharide is conjugated to a different carrier proteinthan the carrier protein to which Streptococcus pneumoniae serotype 6Acapsular polysaccharide is conjugated.
 25. (canceled)
 26. An immunogeniccomposition according to claim 13 comprising serotype 19F conjugated toDiphtheria toxoid.
 27. An immunogenic composition according to claim 13wherein the composition comprises capsular saccharide 18C conjugated totetanus toxoid (TT).
 28. An immunogenic composition according to claim20 further comprising one or more S. pneumoniae capsular polysaccharideof: serotype 33F, serotype 15 and serotype 12F, conjugated to a carrierprotein.
 29. (canceled)
 30. (canceled)
 31. An immunogenic compositionaccording to claim 20 which further comprises an adjuvant, wherein theadjuvant comprises (per 0.5 mL dose) 100-750, 200-500, or 300-400 μg Al(aluminium) as aluminium phosphate.
 32. (canceled)
 33. A vaccinecomprising the immunogenic composition of claim 20 and apharmaceutically acceptable excipient or carrier.
 34. (canceled)
 35. Amethod for the treatment of Streptococcus pneumoniae infection in asubject in need thereof comprising administering to said subject atherapeutically effective amount of an immunogenic composition of claim20 or the vaccine of claim
 33. 36. A method of immunising a human hostagainst Streptococcus pneumoniae infection comprising administering tothe host an immunoprotective dose of the immunogenic composition of anyof claim 20 or vaccine of claim
 33. 37. A method of inducing an immuneresponse to Streptococcus pneumoniae serotype 6A in a subject, themethod comprising administering a therapeutically or prophylacticallyeffective amount of the immunogenic composition of claim 20 or thevaccine of claim
 33. 38. (canceled)
 39. (canceled)